ALBERT

Description: ABSTRACT Signals from growth factors or mechanical stimuli converge to promote vascular smooth muscle cell (VSMC) migration and proliferation, key events in the pathogenesis of intimal hyperplasia upon vascular injury. Spry1, a regulator of receptor tyrosine kinases (RTK), plays a role in maintaining the contractile phenotype of VSMC. The aim of the current study was to determine the role of Spry1 in VSMC proliferation in vitro and injury induced neointimal hyperplasia in vivo. VSMC proliferation and neointima formation were evaluated in cultured human aortic SMC (hAoSMC) and ligation-induced injury of mouse carotid arteries from Spry1 gene targeted mice, and their corresponding wild type littermates. Human Spry1 or non-targeting control lentiviral shRNAs were used to knock down Spry1 in hAoSMC. Time course cell cycle analysis showed a reduced fraction of S-phase cells at 12 and 24 hours after growth medium stimulation in Spry1 shRNA transduced hAoSMC. Consistent with reduced S-phase entry, the induction of cyclinD1 and the levels of pRbS807/S811, pH3Ser10, and pCdc2 were also reduced, while the cell cycle inhibitor p27 Kip1 was maintained in Spry1 knockdown hAoSMC. In vivo, loss of Spry1 attenuated carotid artery ligation-induced neointima formation in mice, and this effect was accompanied by a decrease in cell proliferation similar to the in vitro results. Our findings demonstrate that loss of Spry1 attenuates mitogen-induced VSMC proliferation, and thus injury-induced neointimal hyperplasia likely via insufficient activation of Akt signaling causing decreased cyclinD1 and increased p27 Kip1 and a subsequent decrease in Rb and cdc2 phosphorylation. This article is protected by copyright. All rights reserved

Description: PTPRJ is known for its antiproliferative role. Loss of heterozygosity (LOH) of PTPRJ has frequently been observed in various human cancers including colorectal cancer (CRC), lung cancer and breast cancer. However, the function and mechanism of PTPRJ in CRC is not well understood. At the present study, we show that ectopic expression of PTPRJ inhibits cell growth, migration and invasiveness in CRC cell line HCT116. Moreover, PTPRJ inhibits the tumorigenecity of HCT116 in a xenograft tumor model. MiR-155, the well-known oncomiR in CRC, is identified as an upstream factor of PTPRJ. MiR-155 directly binds to the 3' untranslated region of PTPRJ mRNA and suppresses the mRNA and protein levels of PTPRJ. Furthermore, the growth-promoting and AKT signaling activation effect of miR-155 was abrogated by PTPRJ overexpression, and vice versa. Our study reveals the crucial role of miR-155/PTPRJ/AKT axis in proliferation and migration of CRC cells and suggests a therapeutic potential of PTPRJ.This article is protected by copyright. All rights reserved

Description: Due to increasing of the orthopedic lesions and fractures in the world and limitation of current treatment methods, researchers and surgeons paid attention to the new treatment ways especially to tissue engineering and regenerative medicine. Innovation in stem cells and biomaterials accelerate during the last decade as two main important parts of the tissue engineering. Recently, induced pluripotent stem cells (iPSCs) introduced as cells with highly proliferation and differentiation potentials that hold great promising features for used in tissue engineering and regenerative medicine. As another main part of tissue engineering, synthetic and natural polymers have been shown daily grow up in number to increase and improve the grade of biopolymers that could be used as scaffold with or without stem cells for implantation. One of the developed areas of tissue engineering is bone tissue engineering; the aim of this review is present studies were done in the field of bone tissue engineering while used iPSCs in combination with natural and synthetic biomaterials. This article is protected by copyright. All rights reserved

Description: ABSTRACT Wilms' tumor (WT) is a most common renal cancer that occurs among children, and microRNA-19b (miR-19b) usually participates in various human cancers. Importantly, the PTEN/PI3K/Akt signaling pathway plays a key role in cell apoptosis, growth and proliferation. Thus, our present study aims to investigate the effect of miR-19b on the PTEN/PI3K/Akt signaling pathway during WT cell proliferation, migration and apoptosis. WT tissues and adjacent normal tissues from WT patients were collected. qRT-PCR was applied to detect miR-19b expression in both the WT tissues and the adjacent normal tissues, immunohistochemistry was applied to detect the protein expressions of PTEN, P13K and p-Akt, SK-NEP-1 cells were divided into the blank, negative control (NC), miR-19b mimics and miR-19b inhibitors groups. MTT assay, propidium iodide (PI) staining, Annexin-V/PI double-staining, Transwell assay and Western blotting were performed to examine cell proliferation, cycle, apoptosis, migration and invasion, and the protein expressions of PTEN, P13K, Akt and p-Akt. Increased miR-19b expression, positive expression rates of P13K and Akt, decreased PTEN expression rate, a negative correlation between PTEN expression and tumor lymph node metastasis, and a positive correlation between the expression of P13K and Akt and the clinical stages were observed in the WT tissues. The miR-19b inhibitors group exhibited decreased cell proliferation, cell cycle progression, migration and invasion, and protein expressions of PI3K and p-Akt but increased PTEN protein expression compared with the blank and NC groups. Thus, inhibition of miR-19b suppresses the progression of WT by modulating the PTEN/PI3K/AKT signaling pathway. This article is protected by copyright. All rights reserved

Description: Multi-cause-induced interstitial lung disease, particularly pulmonary fibrosis, is a serious clinical concern. fibroblasts have been suggested to have a major role, with it recently being revealed that some of these fibroblasts are derived from alveolar epithelial cells through epithelial-mesenchymal transition (EMT). Eukaryotic translation initiation factor 3 subunit A (EIF3A) is a protein that in humans is encoded by the EIF3A gene, and has been suggested to play roles in regulating translation of a subset of mRNAs and in regulating cell cycle progression and cell proliferation. In the present study, we chose a well-known TGFβ1-induced EMT model in alveolar epithelial cells to investigate the functional role of EIF3A. TGFβ1 induced EIF3A expression and EMT process in alveolar epithelial cells, after EIF3A knockdown, the EMT process could be partially reversed. Online tools and luciferase assays showed that miR-497 could inhibit EIF3A expression by directly binding to the 3'UTR of EIF3A. Ectopic miR-497 expression partially reversed TGFβ1-induced EMT in alveolar epithelial cells. In addition, miR-497 could suppress TGFβ1-induced pulmonary fibroblast proliferation and EIF3A, Collagen I and α-SMA protein levels. Taken together, EIF3A could promote TGFβ1-induced EMT in alveolar epithelial cells; miR-497 suppressed TGFβ1-induced EMT in alveolar epithelial cells TGFβ1-induced excessive proliferation and ECM in pulmonary fibroblast through inhibiting EIF3A by targeting. MiR-497/EIF3A axis shows the potential to be effective in the treatment of pulmonary fibrosis. This article is protected by copyright. All rights reserved

Description: Signal transducers and activators of transcription 1 (STAT1) exhibits tumor-suppressor properties by inhibiting oncogenic pathways and promoting tumor immunosurveillance. MicroRNAs, a group of non-coding endogenous ones, may regulate gene expression and plays specific roles in tumorigenesis. Recently, miR-181a has been reported to be associated with poor prognosis of colorectal cancer (CRC). Using human colorectal cancer cell lines, we demonstrated that STAT1 suppresses both LoVo and SW480 cell growth by down-regulating miR-181a. STAT1 regulates the expression of miR-181a through binding to the elements in the miR-181a's promoter region. Further, we revealed that miR-181a accelerates CRC cell proliferation through phosphatase and tensin homolog on chromosome ten (PTEN). In addition, PTEN protein was upregulated in response to STAT1 overexpression or miR-181a inhibition, downregulated in response to STAT1 knockdown or miR-181a overexpression. Without changes on the AKT protein level, p-AKT was downregulated by STAT1 overexpression or miR-181a inhibition while upregulated by STAT1 knockdown or miR-181a overexpression, indicating PTEN/Akt pathway activated in STAT1/miR-181a regulation of CRC cell proliferation. Taken together, our findings shed new light on the STAT1/miR-181a/PTEN pathway in colorectal cancer and add new insight regarding the carcinogenesis of colorectal cancer. This article is protected by copyright. All rights reserved

Description: ABSTRACT The present study investigated the action of copaiba oil ( Copaifera reticulata ) on the systemic inflammation, oxidative status and liver cell metabolism of rats with adjuvant-induced arthritis. The later is an experimental autoimmune pathology that shares many features with the human rheumatoid arthritis. Holtzman rats were distributed into the following groups: control (healthy) rats; control rats treated with copaiba oil at the doses of 0.58 and 1.15 g · Kg −1 , arthritic rats, and arthritic rats treated with copaiba oil (0.58 and 1.15 g · Kg −1 ). The oil was administrated orally once a day during 18 days after arthritis induction. Both doses of copaiba oil improved the paw edema and the dose of 0.58 mg · Kg −1 improved the swollen adrenals and lymph nodes besides decreasing the plasmatic myeloperoxidase activity (-30%) of arthritic rats. Copaiba oil (1.15 g · Kg −1 ) abolished the increases of protein carbonyl groups and reactive oxygen species in the liver and both doses increased the liver GSH content and the catalase activity in arthritic rats. Copaiba oil (1.15 g · Kg −1 ) decreased glycolysis (-65%), glycogenolysis (-58%) and gluconeogenesis (-30%) in the liver of arthritic animals. However, gluconeogenesis was also diminished by the treatment of control rats, which presented lower body weight gain (-45%) and diminished number of hepatocytes per liver area (-20%) associated to higher liver weight (+29%) and increased hepatocyte area (+13%). The results reveal that copaiba oil presented systemic anti-inflammatory and antioxidant actions in arthritic rats. These beneficial effects, however, were counterbalanced by harmful modifications in the liver cell metabolism and morphology of healthy control rats. This article is protected by copyright. All rights reserved

Description: ABSTRACT Recent studies have demonstrated the influential role of microbial stimulus in characteristics and immunomodulatory effects of mesenchymal stem cells (MSCs). Due to the migration of MSCs to infection site, it is of importance to understand the interaction of microbial ligands with MSCs in order to clarify the positive or negative role of MSCs in the control of infection. In this research, we assess leishmanial soluble antigen (LSA)-primed MSCs on macrophage immune responses to lipopolysaccharide (LPS). For this purpose, the effects of both conditioned media (CM) and cell-cell contact of L SA primed MSCs were determined on macrophage responses to LPS. According to the obtained results, MSC-treated macrophages demonstrated an alternatively activated macrophages with higher levels of interleukin-10 (IL-10) and transforming growth factor-alpha (TNF-α) and lower levels of IL-6 and nitric oxide (NO) production as compared to the controls. In addition, phagocytosis of apoptotic thymocytes was induced in MSC-treated macrophages. In conclusion, it seems that MSCs trigger an anti-inflammatory phenotype in macrophages at Leishmania infected sites in order to enhance the induction of immune regulatory cells and clearance of apoptotic cells. This article is protected by copyright. All rights reserved

Description: Background Urinary stone disease (USD) is increasing in adult and pediatric populations Adult and pediatric studies have demonstrated decreased bone mineral density and increased fracture rates. USD has also been independently linked to increased rates of myocardial infarction and cerebral vascular accidents. Although USD is a multisystem disorder involving the kidneys, bone and vasculature, the molecular mechanisms linking these three organs remain unknown. Methods Calcium oxalate nephropathy was induced in C57BL/6J mice with intra-peritoneal (ip) injection of sodium glyoxolate. Half of each kidney underwent Pizzalato staining and half was snap frozen for RNA extraction. RT2 Profiler Mouse Atherosclerosis, Osteoporosis and Calcium Signaling PCR Arrays (Qiagen) were performed. Only results that passed quality checks in PCR array reproducibility and genomic DNA contamination were included. Genes had to show at least 4 fold differential expression and p 〈0.01 to be considered significant. Results Atherosclerosis Array showed upregulation of 19 genes by four fold, 10 of which were ≥10 fold. All 19 had p ≤0.002. The Osteoporosis Array showed fourfold upregulation of 10 genes, 5 showed 〉10 fold increase. All 10 have p≤0.003. The Calcium Signaling Array showed significant fourfold upregulation of 10 genes, four of which were ≥10 fold. All 10 have p≤0.03. Conclusion We have demonstrated that calcium oxalate nephropathy can induce upregulation of atherosclerotic, metabolic bone and calcium homeostasis genes in a murine model. This may be and initial step in identifying the molecular mechanisms linking stone, bone and cardiovascular disease. This article is protected by copyright. All rights reserved

Description: Objectives : To gain insight into the effect of metformin on losing weight from peptidomic perspective and to screen potential active peptides for reducing fat lipid deposition. Methods : After determining the proper concentration of metformin on human primary visceral adipocytes, we constructed a comparative peptidomic profiling between control and metformin treatment group (n = 3) using a stable isobaric labeling strategy involving tandem mass tag reagents, followed by liquid chromatography tandem mass spectrometry. Results : We identified and quantified 3065 non-redundant peptides, 304 of which were differentially expressed after metformin treatment, 206 peptides were up regulated and 98 peptides were down regulated significantly. Gene ontology (GO) enrichment and pathway analysis were performed to study differentially peptides though their precursor proteins. Conclusions : We concluded three peptides located within the functional domains of their precursor proteins could be candidate bioactive peptides for obesity. On one hand, these results confirmed the versatile effects of metformin on adipocyte and advance our current understanding of metformin, on the other hand, these identified peptides might play putative roles in treatment of obesity. This article is protected by copyright. All rights reserved

Description: The study aims to analyze the key signaling pathways in regulating the process of embryonic stem cells (ESCs) differentiation into spermatogonial stem cells (SSCs). We explored the specific regulating mechanisms of C-Jun amino-terminal kinase (JNK) signaling in this process. Interference/overexpression of MAPK8 allows the JNK signaling pathway to be blocked/activated. In Retinoic acid (RA) induced in vitro differentiation assays, the expression of germ cell marker genes, cvh, c-kit, integrin α6 and integrin β1 , was observed to upregulate while activating JNK signaling significantly. Fluorescence Activated Cell Sorting (FACs) analysis showed that the proportion of cvh + and integrin α6 + cells in the overexpression group was significantly higher than which in the RA + shRNA- MAPK8 group. In in vivo situations, shRNA- MAPK8 could stably express in chicken embryos and significantly down-regulate expression of MAPK8 and downstream genes in JNK signaling pathway. With PAS stain, we found that PGCs(primordial germ cells) was significantly decreased after inhibiting MAPK8 . With real-time quantitative PCR (qRT-PCR) and Western Blot, we identified that reproductive related genes expression was significantly suppressed after inhibiting MAPK8 in vivo . We preliminarily concluded that knockdown/ overexpression of MAPK8 could affect differentiation of ESC by inhibiting/activating JNK signal. This article is protected by copyright. All rights reserved

Description: Objective: This study is designed to determine whether lincRNA-DYNLRB2-2 could promote cholesterol efflux through regulating the expression of TLR2. Methods: THP-1 and RAW264.7 cells were incubated with oxLDL for 48 h to induce the formation of foam cells, and ORO staining was performed and intracellular cholesterol contents were measured by HPLC assay. qRT-PCR and Western blotting were performed to detect mRNA and protein expression levels, respectively. Lentiviral vector LV-DYNLRB2-2 and lincRNA-DYNLRB2-2 siRNA was constructed to explore its potential role. The cholesterol efflux was assessed by liquid scintillation counting. The effects of TRL2 were determined in apoE −/− mice that fed a high fat diet and were randomly divided into three groups and infected with LV-Mock, LV-Sh- TRL2 or LV- TRL2. Atherosclerosis was observed in the aortic sinus and the levels of cytokines and serum biochemical parameters were measured. Results: Ox-LDL induced foam cell formation in the THP-1 and RAW264.7 cells. LincRNA DYN-LRB2-2 was upregulated in oxLDL –treated THP-1 and Raw264.7 cells. LincRNA-DYNLRB2-2 plays important role in regulating the cholesterol efflux, ABCA1 expression level and anti-inflammatory processes in THP-1 and RAW264.7 cells. Further study indicated that lincRNA-DYNLRB2-2 negatively regulated TRL2 expression and TRL2 overexpression reversed the effects of lincRNA-DYNLRB2-2 on cholesterol efflux and ABCA1 expression level in THP-1 and RAW264.7 cells. Besides, we found TRL2 plays important role in lipid accumulation, plaque formation and regulating serum inflammatory cytokines level in apoE −/− mice with a high fat diet. Conclusion: LincRNA DYN-LRB2-2 upregulates cholesterol efflux by decreasing TLR2 expression in macrophages. This article is protected by copyright. All rights reserved

Description: Objectives: The purpose of the present study was to investigate the possible therapeutic effects of the human Wharton-Jelly mesenchymal stromal cells derived micro-vesicles (hWJMSCs-MVs) on renal ischemia-reperfusion injury (IRI) after cardiac death (CD) renal transplantation in rats. Methods: MVs were injected intravenously in rats immediately after renal transplantation. The animals were sacrificed at 24 h, 48 h, 1 and 2 weeks post-transplantation. ELISA was used to determine the von Willebrand Factor (vWF), tumore necrosis factor (TNF)-α and interleukin (IL)-10 levels in the serum. Tubular cell proliferation and apoptosis were identified by Ki67 immunostaining and TUNEL assay. Renal fibrosis was assessed by Masson's tri-chrome straining and alpha-smooth muscle actin (α-SMA) staining. The infiltration of inflammatory cells was detected by CD68 + staining. The transforming growth factor (TGF)-β, hepatocyte growth factor (HGF) and α-SMA expression in the kidney was measured by western blot. Results: After renal transplantation, the rats treated with hWJMSCs-MVs improved survival rate and renal function. Moreover, MVs mitigated renal cell apoptosis, enhanced proliferation, and alleviated inflammation at the first 48 h. In the late period, abrogation of renal fibrosis was observed in the MVs group. MVs also could decrease the number of CD68 + macrophages in the kidney. Furthermore, MVs decreased the protein expression levels of α-SMA and TGF-β1 and increased the protein expression level of HGF at any point (24 h, 48 h, 1 or 2 weeks). Conclusion: The administration of MVs immediately after renal transplantation could ameliorate IRI in both the acute and chronic stage. This article is protected by copyright. All rights reserved

Description: ABSTRACT Increasing evidence from various clinical and experimental studies has demonstrated that the inflammatory microenvironment created by immune cells facilitates tumor migration. Epithelial-mesenchymal transition (EMT) is involved in the progression of cancer invasion and metastasis in an inflammatory microenvironment. B-lymphoma Moloney murine leukemia virus insertion region 1 (BMI-1) acts as an oncogene in various tumors. Ectopic expression of Bmi-1 have an effect on EMT and invasiveness. The purpose of this study was to investigate the efficacy of BMI-1 on inflammation-induced tumor migration and EMT and the underlying mechanism. We observed that the expression of BMI-1, TNF-α and IL-1β was significantly increased in HT29 and HCT116 cells after THP-1 Conditioned-Medium (THP-1-CM) stimulation. Additionally, inhibition of BMI-1 impeded cell invasion induced by THP-1-CM-stimulation in both HT29 and HCT116 cells. BMI-1 knockdown remarkably repressed THP-1-CM -induced EMT by regulating the expression of EMT biomarkers with an increase in E-cadherin accompanied by decrease in N-cadherin and vimentin. Furthermore, downregulation of BMI-1 dramatically impeded THP-1-CM-triggered Toll-like receptor 4(TLR4)/myeloid differentiation protein 2(MD-2)/ myeloid differentiation factor 88(MyD88) activity by repressing the expression of the TLR4/MD-2 complex and MyD88. Further data demonstrated that knockout of BMI-1 also dampened NF-κB THP-1-CM-triggered activity. Taken all data together, our findings established that BMI-1 modulated TLR4/MD-2/MyD88 complex-mediated NF-κB signaling involved in inflammation-induced cancer cells invasion and EMT, and therefore could be a potential chemopreventive agent against inflammation-associated colorectal cancer. This article is protected by copyright. All rights reserved

Description: Urothelial carcinoma associated 1α (UCA1α) is a novel long non-coding RNA (lncRNA) that regulates bladder cancer proliferation, migration, and invasion. The target genes of UCA1α have, however, not been identified. To address this, a pCDNA3.1(+)-UCA1α over-expression vector was transfected into UM-UC-2 bladder cancer cells. Genes differentially expressed between pCDNA3.1(+)-UCA1α and pCDNA3.1(+) transfected cell were then detected by microarray and bioinformatics analysis. A total of seventy-one differentially expressed genes were identified, including fifty-two up-regulated genes and nineteen down-regulated genes. As expected, the lncRNA UCA1α expression level was significantly increased when compared to that of pCDNA3.1(+) transfected cells. The five most significantly up-regulated and five most significantly down-regulated genes were selected, and their expression levels were also assessed by real time quantitative polymerase chain reaction and western blot. The mRNA and protein expression levels of FOXI3 and GSTA3 were found to be significantly increased, and those of MED18 and TEX101 were found to be significantly decreased. Gene ontology (GO) clustering identified several significant biological processes, cellular components, and molecular functions, associated with lncRNA UCA1α over-expression. The differentially expressed genes were involved in several significant pathways as shown by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway clustering. Cell proliferation activity was significantly increased following overexpression of lncRNA UCA1α increasing over culture time. The present study identifies, for the first time, potential target genes for lncRNA UCA1α in bladder cancer, and provides a significant reference for studying the role of lncRNA UCA1α in bladder cancer. This article is protected by copyright. All rights reserved

Description: ABSTRACT Migration of skeletal muscle precursor cells is required for limb muscle development and skeletal muscle repair. This study aimed to examine the role of P2Y6 receptor in C2C12 myoblasts migration. C2C12 myoblasts were treated with P2Y6 agonist UDP, P2Y6 antagonist MRS2578, Ca 2+ channel blocker BTP2, or ROCK inhibitor GSK269962 or Y27632, and the migration ability of C2C12 cells was assessed by wound healing assay. The cellular Ca 2+ content was analyzed with fluo-4 probe and the activation of ROCK (phosphorlyation of LIMK and cofilin) was assayed by western blot. The cytoskeleton was labeled with Actin-Tracker Green and Tubulin-Tracker-Red. Silencing P2Y6 expression in C2C12 myoblasts reduced intracellular Ca 2+ content and cell motility. Whereas UDP increased cellular Ca 2+ content, actin filaments, and cell migration, MRS2578 had the opposite effects. The effects of UDP were abrogated by BTP2 and GSK269962 (and Y27632). Disruption of P2Y6 signaling pathway caused C2C12 myoblasts to have an elongated morphology. These results demonstrated that P2Y6 signaled through Ca 2+ influx and RhoA/ROCK to reorganize cytoskeleton and promote migration in myoblasts. This article is protected by copyright. All rights reserved

Description: Cancer cytokines are known to mediate several complex cancer cell physiologies. Also, cancer cells themselves are known to secrete cytokines whose expressions and net inducible results are controlled by a variety of factors. We profiled a few cytokines secreted by 2D, 3D aggregates and the 3DRs of MCF-7 cell line at various time points. Several cytokines were seen more expressed by 3D cultures on the 4 th day and IL-10 peaked on the day 1 of 3D cultures while TNF-α level peaked on the 7 th day. α-Defensin, SDF-7 and TGF-β also showed markedly higher levels. There was a reduced expression of IL-6 and IL-17 by the 3DRs. TGF-β did not show much change among the 2D, progressive 3D and 3DR cultures. Utilizing 3DRs as study material will be a significant extension of the ways cells lines can be used for research. This article is protected by copyright. All rights reserved

Description: ABSTRACT microRNA-145 ( miR-145 ) has been shown to act as a tumour suppressor in colorectal cancer but its role in the regulation of epithelial-mesenchymal transition (EMT) is unclear. Ectopic expression of miR-145 suppressed the proliferation, migration and invasion in SW480 but surprisingly enhanced these traits in its metastatic counterpart, SW620 cells while anti miR-145 reversed the effects of miR-145 in both of these human colorectal cancer cells. In SW480 and SW620 cells, Smad -interacting protein 1 ( SIP1 ), was identified as a target of miR-145 , and its expression was suppressed both at mRNA and protein levels and siRNA- SIP1 mimicked the effects of miR-145 . Further, re-introduction of SIP1 alone or its co-expression with miR-145 , rescued SW480 and SW620 cells from the effects of miR-145 , indicating that the distinct functions of miR-145 might be mediated, in part, through SIP1 . Since Wnt signalling plays an essential role in EMT in CRC progression, the effects of miR-145 on the expression of Wnt signalling intermediates and EMT markers were studied. Re-expression of miR-145 was found to downregulate the expression of CTNNB1, TCF4, CCND1, VIM and SNAI, but, upregulate CDH1 expression in SW480 cells. On the other hand, miR-145 exhibited an oncogenic potential in SW620 cells by actuating Wnt signalling and the expression of EMT-relevant markers. These results strongly hint that the paradoxical functions of miR-145 in the regulation of proliferation, migration and invasion might be mediated through downregulation of SIP1 , and differential tuning of Wnt signalling and EMT-mediators. This article is protected by copyright. All rights reserved

Description: Most studies have revealed the effects of caveolins in cancer inhibition. However, due to a lack of reports about their new transcripts, their presence and their effects on different cancers are unclear. This study was conducted to evaluate the cavolin-2(cav-2) transcripts expression changes in tumoral and corresponding tissues and in contralateral breast, to investigate their variation associated with the variation of caveolin-1 (cav-1) expression in breast cancer. There were 40 breast-derived tumoral, corresponding and contralateral tissues obtained from the patients with breast cancer. The RNA and proteins were extracted from these samples. So, cav-1 and cav-2 transcripts' variation were assessed in whole tumoral, corresponding and contralateral breast. Also, their expression modifications were evaluated via the western blotting technique. The results derived from this study verified the presence of transcript III of cav-2 for the first time, which was reported only in the gene bank, but we could not detect and validate any protein associated with these transcripts. Also, the decreasing trend of cav-1 and the cav-2 (transcripts I and II) were observed in tumoral tissues compared to unaffected tissues especially in stages one and two. It seems that the descending expression levels of cav-1 and cav-2 (transcript I, II) besides the lasting expression of cav-2 (transcript III) are associated with the incidence and promotion of breast cancer, especially in the initial stages of breast cancer. So, this may show a potential in determining the patients who can undergo the prophylactic mastectomy. Moreover, the results of the study demonstrated that transcript III may be a candidate as a non-coding RNA. This article is protected by copyright. All rights reserved

Description: ABSTRACT Our study aimed to analyze the effect of ouabain administration on lipopolysaccharide (LPS)-induced changes in oxidative parameters, membrane lipid composition, and the activities of some important enzymes of the nervous system. The content of phospholipids, cholesterol and gangliosides were analyzed in Wistar rats after intraperitoneal injection of ouabain(1.8 µg/kg), LPS(200 µg/kg) or saline. Oxidative parameters were also evaluated, including the activities of superoxide dismutase, catalase and glutathione peroxidase, the levels of glutathione and lipid peroxidation, as well as Na,K-ATPase activity and the level of glutamate transporter EAAT4. Administration of LPS resulted in increased oxidative stress, as evidenced by an increase in lipid peroxidation levels, glutathione peroxidase activity, decreased catalase activity and reduced glutathione levels. All changes recorded were attenuated by pretreatment with ouabain. Administration of ouabain plus LPS enhanced the total ganglioside content and EAAT4 levels, but failed to alter the Na,K-ATPase activity. Our data suggest a neuroprotective effect of ouabain against LPS-induced oxidative stress by promoting membrane lipid remodeling and increasing the expression of glutamate transporter EAAT4. Our results emphasize that the observed oxidative stress is not correlated with Na,K-ATPase, but with a possible ouabain-mediated effect on cellular signaling. The relevance of our results extends beyond LPS-induced changes in oxidative parameters, as nanomolar doses of ouabain might prove useful in neurodegenerative models. Further study of other cardenolides and related molecules, as well as the development of new molecules derived from ouabain, could also prove useful in the fight against the oxidative and/or general cell stress triggered by neuronal pathologies. This article is protected by copyright. All rights reserved

Description: ABSTRACT This study aims to explore the role of long noncoding RNA (lncRNA)-HOX transcript antisense intergenic RNA (HOTAIR) in the occurrence and progression of glioma. Fresh glioma and normal brain tissues were classified into a glioma group (n = 67) and a normal group (n = 64) respectively. U87 cells were assigned into the blank, sh-NC and sh-HOTAIR groups. Quantitative real-time polymerase chain reaction (qRT-PCR) was utilized to determine HOTAIR expression. Cell proliferation, cell cycle and cell apoptosis rates were detected by cell counting kit-8 (CCK-8) and flow cytometry (FCM). Scratch test and transwell assay were conducted for cell migration and invasion. Orthotopic glioma tumor model in nude mice was established by inoculating tumor cell suspension. Hematoxylin-Eosin (HE) staining was used to observe the growth and invasion of orthotopic glioma tumors. The expression of HOTAIR and cell viability was found to be lowest in the sh-HOTAIR group among the three groups. The sh-HOTAIR group exhibited a higher apoptotic rate and lower number of cell migration compared with the blank and sh-NC groups. Additionally, the speed of wound healing was slower, the migration distance decreased and the survival time of nude mice was extended in the sh-HOTAIR compared to the other groups. Moreover, the sh-HOTAIR group demonstrated reduced lesion sizes and inflammation, no convulsions or hemiplegia and lesser number of satellite metastases. Our findings support that down-regulation of HOTAIR could inhibit cell proliferation, promote cell apoptosis as well as suppress cell invasion and migration in the progression of glioma. This article is protected by copyright. All rights reserved

Description: The aim of this study was to evaluate the toll like signaling pathway and atrophy after sleep deprivation (SD) in rat masticatory muscles: masseter and temporal. A total of twenty-four animals was distributed into three groups: Control group (CTL, n = 8), subjected to SD for 96 hours (SD96, n = 8) and subjected to SD for 96 hours more 96 hours of sleep recovery (SD96 + R, n = 8). Histopathological analysis revealed the presence of acute inflammatory cells, congested vessels, fibrosis and high cellularity in the skeletal muscle fibers from masseter and temporal submitted to SD. These morphological alterations were not observed in the control group since neither inflammatory cells nor congested vessels were observed to this group. In the group SD96 + R, the absence of inflammation was noticed to the masseter only. In this group, COX-2 and TNF-alpha downregulation were detected when comparing to control group. MyD88 and pIKK decreased in SD96 and SD96+ R groups being pNFKBp50 downregulatated in SD96 + R. MyD88 expression increased in rats submitted to SD96 and SD96 + R in temporal when compared to control group. On the other hand, pIKK decreased the protein expression in groups SD96 and SD96 + R while pNFKBp50 showed a decreased protein expression in group SD96 only. The activation of atrophy by means of MAFbx upregulation was detected in temporal muscle in SD96 and SD96 + R when compared to control. In summary, our results show that SD is able to induce morphological alterations in rat masticatory muscles. Toll like signaling pathway and atrophy play important roles in ethiopathogenesis induced by SD, being dependent of skeletal muscle type. This article is protected by copyright. All rights reserved

Description: In the pathological mechanism of pulmonary arterial hypertension, the role of 31 apoptosis-resistant pulmonary microvascular endothelial cells (PVECs/ AR) has been 32 emphasized on the pulmonary vascular remodeling. In the present study, we investigated 33 whether PVECs/ AR can promote the proliferation and migration of pulmonary arterial 34 smooth muscle cells (PASMCs), and to study the role of miR-195-5p in the crosstalk between 35 these two types of cells. We confirmed that PVECs/ AR can promote the proliferation and 36 migration of PASMCs in a co-culture system of AR/ PVECs and PASMCs. Additionally, 37 after exposure to hypoxia for 12 or 24 hours, AR/ PVECs had a higher mature miR-195-5p 38 level than PVECs (P 〈 0.05, 12 h and 24 h). Luciferase reporter assays were used to validate 39 indications of the existence of an HRE in the miR-195-5p promoter. Knocking down Smad7 40 can reverse the inhibition of Lv-S195 on TGF-β1-induced PASMCs remodeling. TGF-β1 41 promoted cell growth in PASMCs, and the supernatant of PVECs/AP infected with Lv-S195 42 inhibited TGF-β1 enhanced proliferation in PASMCs, which was also blocked by 43 Lv-shRNA-Smad7. The result of this experiment confirmed the specificity of the 44 HIF-1a/miR-195/Smad7 pathway. Our data indicate the possible function of PVECs/ AR in 45 the process of pulmonary vascular remodeling. MiRNA-195-5p played a role as an interacting 46 paracrine factor between PVECs/ AR and PASMC, which seemed to function through the 47 HIF-1a/miRNA-195-5P /Smad7 pathway. This article is protected by copyright. All rights reserved

Description: Pancreatic cancer (PC) has a high mortality rate in all cancers worldwide. According to recent studies, long non-coding RNA-CASC2 is involved in the development and progression of many malignant tumors; in the present study, we demonstrated that lncRNA-CASC2 was specifically downregulated in PC tissues and cell lines, and a lower CASC2 expression in PC was related with a poorer prognosis. CASC2 suppressed PC cell proliferation. Hepatocyte nuclear factor 1 alpha (HNF1A) is a transcription factor known to regulate pancreatic differentiation and maintain the homeostasis of endocrine pancreas. Recently, HNF1A is considered to be a possible tumor suppressor in PC. In the present study, we observed that HNF1A positively regulated CASC2 expression. Through Luciferase assays, we demonstrated that CASC2 gene possessed a HNF1A-responsive element (CASC2-HNF1A RE); HNF1A could promote CASC2 expression through direct binding to CASC2-HNF1A RE. Further, PTEN/Akt signaling was involved in HNF1A regulation of CASC2. Finally, we evaluated the expression level of HNF1A in PC tissues; lower HNF1A expression was correlated with shorter overall survival (OS) in patients with PC. Taken together, these findings will shed light to the role and mechanism of HNF1A/CASC2 in regulating PC cells proliferation through PTEN/Akt signaling. CASC2 may serve as a potential therapeutic target in PC in the future. This article is protected by copyright. All rights reserved

Description: Acute promyelocytic leukemia (APL) is one of the most life-threatening hematological malignancies. Defects in the cell growth and apoptotic pathways are responsible for both disease pathogenesis and treatment resistance. Therefore, pro-apoptotic agents are potential candidates for APL treatment. Kaempferol is a flavonoid with antioxidant and anti-tumor properties. This study was designed to investigate the cytotoxic, pro-apoptotic and differentiation-inducing effects of kaempferol on HL-60 and NB4 leukemia cells. Resazurin assay was used to determine cell viability following treatment with kaempferol (12.5-100 µM) and all- trans retinoic acid (ATRA; 10 µM; used as a positive control). Apoptosis and differentiation were also detected using propidium iodide and NBT staining techniques, respectively. Furthermore, the expression levels of genes involved in apoptosis (PI3K, AKT, BCL2, BAX, p53, p21, PTEN, CASP3, CASP8 and CASP9), differentiation (PML-RAR and HDAC1), and multi-drug resistance (ABCB1 and ABCC1) were determined using quantitative real-time PCR. The protein expressions of Bax/Bcl2 and casp3 were confirmed using western blot. The results showed that kaempferol decreased cell viability and increased subG1 population in the tested leukemic cells. This effect was associated with decreased expression of Akt, BCL2, ABCB1 and ABCC1 genes, while the expression of CASP3 and BAX/BCL-2 ratio were significantly increased at both gene and protein levels. Kaempferol promoted apoptosis and inhibited multidrug resistance in a concentration-dependent manner, without any differential effect on leukemic cells. In conclusion, this study suggested that kaempferol may be utilized as an appropriate alternative for ATRA in APL patients. This article is protected by copyright. All rights reserved

Description: Spinal cord injury (SCI) is lead to locomotor impairment because of neurological damage after following trauma. Quercetin (Que) has been confirmed have a neuro-protective effect during nerve damage processes. The purpose of this study was to determine the roles of Que in functional recovery, cavity formation, astrocyte activation and nerve regeneration following SCI. Sprague-Dawley rats were randomly divided into 3 groups: Sham group, SCI group and Que + SCI group. A rat model of SCI was made at T10 using the modified Allen's method. In the Que + SCI group, animals underwent laminectomy and were then intraperitoneally injected with 20 mg/kg Que for 7 days. Locomotor function was determined with the Basso, Beattie, Bresnahan (BBB) scores at 1, 3, 5 and 7 days post injury. At 7 days post injury, somatosensory evoked potentials (SEPs) and motor evoked potentials (MEPs) were recorded. Hematoxylin-Eosin (HE) staining was used to investigate cavity formation. Astrocyte activation was assayed by immunohistochemistry staining with an antibody specific for glial fibrillary acidic protein (GFAP), as well as the expression of GFAP and S100β. Axons were stained using an antibody specific for neurofilament 200 (NF200) and 5-hydroxytryptamine (5-HT). In addition, the protein level of BDNF, p-JNK2 and p-STAT3 was detected using western blot. Que promoted locomotor function and electrophysiological recovery, reduced cavity formation, contributed to astrocyte activation and axonal regeneration after acute SCI. Moreover, Que up-regulated the expression of BDNF, but reduced p-JNK2 and p-STAT3 expression after acute SCI. Taken together, Que promoted locomotor and electrophysiological functional recovery, astrocyte activation and axonal regeneration after acute SCI, possibly through BDNF and JAK2/STAT3 signaling pathways. This article is protected by copyright. All rights reserved

Description: ABSTRACT This study aims to investigate the effects of Cyclin D1 silencing on cell cycle, cell proliferation and apoptosis of hepatocellular carcinoma cells (HCC). Cells were divided into the blank group, negative control group (HCC cells transfected with control shRNA), Cyclin D1 shRNA group (HCC cells transfected with Cyclin D1 shRNA) and the normal group (human normal liver L-02 cells). Expressions of Cyclin D1, Caspase-3, Bcl-2 and C-myc were detected by RT-qPCR and Western blotting. Cell proliferation was detected by Cell Counting Kit-8. Cell cycle and apoptosis were detected by flow cytometry. Tumor xenograft in nude mice was performed to detect in vivo tumorigenesis. HCC tissues and HCC cells exhibited elevated expression levels of Cyclin D1. Cyclin D1 expression levels was found to be correlated with tumor size and tumor staging.Compared with the normal group, the blank group showed enhanced cell proliferation, a reduction in the amount of cells in G0/G1 phase, increased number cells in S and G2/M phase, reduced apoptosis, elevated expressions of Cyclin D1, Bcl-2 and C-myc, decreased Caspase-3 activity and significant tumorigenicity. In comparison with the blank group, the Cyclin D1 shRNA group revealed weakened cell proliferation, reduced cells in S and G2/M phase, increased cells in G0/G1 phase, increased Annexin V positive cell ratio, decreased expression of Cyclin D1, Bcl-2 and C-myc, elevated Caspase-3 activity and inhibited tumorigenicity. In conclusion, Cyclin D1 gene silencing suppresses cell proliferation and inhibits cell apoptosis, which may be a new target approach in the treatment and management for HCC.This article is protected by copyright. All rights reserved

Description: Long-term use of methamphetamine (MA) causes a broad range of cognitive deficits. Recently, it has been reported insulin signaling and mitochondrial biogenesis are involved in cognitive processes. This study aimed to examine whether MA induces cognitive deficits concomitant with insulin signaling impairment and mitochondrial dysfunctions and also intranasal (IN) insulin treatment can reverse cognitive deficits caused by MA. Rats were repeatedly treated with increasing doses of MA (1-10 mg/kg) twice a day for 10 days, and their cognitive functions were assessed using Y-maze, novel object recognition and passive avoidance tasks. The expression of components involved in insulin signaling (IR/IRS2/PI3K/Akt/GSK3β) and mitochondrial biogenesis (PGC-1α, NRF1 and TFAM) was measured in the hippocampus. Therapeutic effects of IN insulin delivery (0.5IU/day, for 7 days after MA discontinuation) were also investigated in MA-treated animals. Our results showed that repeated MA exposure induced cognitive deficits, and led to insulin signaling impairment and mitochondrial dysfunction. Interestingly, IN insulin treatment reduced MA-induced cognitive impairments possibly through activating insulin signaling, particularly PI3K/Akt/GSK3β pathway, and mitochondrial biogenesis. Thus, insulin and insulin signaling pathway can be considered as useful targets for the treatment of abnormalities associated with MA abuse. This article is protected by copyright. All rights reserved

Description: ABSTRACT Mitochondrial dysfunction has emerged as a critical pathophysiological factor of myocardial ischemia/reperfusion (I/R) injury. A thorough understanding of mitochondrial dysfunction during I/R at the molecular level is urgently needed. One prominent microRNA, miR-410, was previously reported to be dynamically regulated in diverse cardiomyopathies, but its mechanism is unclear. In the present study, in a cardiac I/R injury mice model, the expression of miR-410 was significantly upregulated, accompanied with decreased mitochondrial function and mitophagy deficit. After an unbiased search for downstream messenger RNA targets of miR-410, effects of the target gene in mitochondrial dysfunction during I/R injury and the underlying mechanism were further explored in cultured human adult cardiac myocytes (HACMs). The results showed that MitoTracker Red-labeled HACMs mitochondria overlapped with GFP-LC3-labeled autophagosomes, suggesting the presence of mitophagy. MiR-410 expression was significantly increased in hypoxia/reoxygenation (H/R)-stimulated HACMs. MiR-410 overexpression further inhibited cell viability, ATP production, mitochondrial membrane potential and mitophagy level, and increased caspase-3 activity, Bax expression and cytochrome c release. Conversely, inhibition of miR-410 attenuated these effects. We found that miR-410 directly interacted with the 3'-untranslated region of the suppressor of high-mobility group box 1 protein (HMGB1) by Dual-Luciferase assay. Moreover, pcDNA3.1-HMGB1 pretreatment effectively reduced the inhibition effects of cell viability and mitophagy brought by H/R, while all those effects can be attenuated by pretreatment with HSPB1 siRNA transfection. Taken together, our results suggest that miR-410 may inhibit mitophagy after cardiac I/R injury by modulating HSPB1 activity via directly targeting HMGB1. This article is protected by copyright. All rights reserved

Description: PBMCs are essential for immunity and involved in various diseases. To identify genetic variations contributing to PBMCs transcriptome-wide gene expression, we performed a genome-wide eQTL analysis by using genome-wide SNPs data and transcriptome-wide mRNA expression data. To assess whether there are common regulation patterns shared among different tissues/organs, public datasets were utilized to identify common eQTLs shared with PBMCs in lymphoblastoid, monocytes, liver, and brain. Allelic expression imbalance (AEI) assay was employed to validate representative eQTLs identified. We identified 443 cis- and 2,386 trans- eSNPs (FDR 〈 0.05), which regulated 128 and 635 target genes, respectively. A transcriptome-wide expression regulation network was constructed, highlighting the importance of 28 pleiotropic eSNPs and 18 dually (cis- and trans-) regulated genes. Three genes, i.e., TIPRL, HSPB8 and EGLN3, were commonly regulated by hundreds of eSNPs and constituted a very complex interaction network. Strikingly, the missense SNP rs371513 trans- regulated 25 target genes, which were functionally related to poly(A) RNA binding. Among 8,904 eQTLs (p 〈 0.001) identified herein in PBMCs, a minority (163) was overlapped with lymphoblastoid, monocytes, liver, and/or brain. Besides, two cis- eSNPs in PBMC were confirmed by AEI. The present results demonstrated a comprehensive expression regulation network for human PBMCs and may provide novel insights into the pathogenesis of immunological diseases related to PBMCs. This article is protected by copyright. All rights reserved

Description: ABSTRACT We aim to investigate the interaction between the EZH2 and the long noncoding RNA (lncRNA) SPRY4-IT1. We also explore their respective effects on human lung adenocarcinoma (LA) cell invasion and migration. Both LA and adjacent normal tissues were obtained from 256 LA patients. SPTY4-IT expression and EZH2 mRNA expressions in tissues and cells were detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR). The siRNAs against SPRY4-IT1 and EZH2 were co-transfected into A549 and H1975 cells. The interaction between SPRY4-IT1 and EZH2 was determined using a RNA pull-down assay and a RNA immunoprecipitation (RIP) assay. A Transwell assay and scratch assay were used to evaluate the cell migration and invasion abilities. The expressions of E-cadherin and Vimentin in the epithelial-mesenchymal transition (EMT) and EZH2 protein expression were detected through western blotting. SPRY4-IT1 expression was observed to be significantly lower, while the expression of EZH2 was higher in the LA tissues than in the adjacent normal tissues. Compared with the HBE cell line, expressions of SPRY4-IT1 in each human LA cell line had decreased, with the lowest observed reduction in the A549 cell line, while EZH2 mRNA and protein expression increased in each human LA cell lines. After SPRY4-IT1-siRNA was transfected into A549 and H1975 cells, invasion and migration abilities were enhanced, in addition to a reduction in the expression of E-cadherin, while expressions of Vimentin exhibited an increased rate. Consequently, we find that EZH2 promotes LA cell invasion and metastasis by inhibiting SPRY4-IT1 expression. This article is protected by copyright. All rights reserved

Description: ABSTRACT Acute myelogenous leukemia (AML) 1 is an aggressive hematologic cancer characterized by infiltration of proliferative, clonal, abnormally differentiated cells of myeloid lineage in the bone marrow and blood. Malignant cells in AML often exhibit chromosomal and other genetic or epigenetic abnormalities that are useful in prognostic risk assessment. In this study, the relative expression and novel single-stranded DNA (ssDNA) binding function of purine-rich element binding proteins A and B (Purα and Purβ) were systematically evaluated in established leukemia cell lines and in lineage committed myeloid cells isolated from patients diagnosed with a hematologic malignancy. Western blotting revealed that the Purα and Purβ are markedly elevated in CD33 + /CD66b + cells from AML patients compared to healthy subjects and to patients with other types of myeloid cell disorders. Results of in silico database analysis of PURA and PURB mRNA expression during hematopoiesis in conjunction with the quantitative immunoassay of the ssDNA-binding activities of Purα and Purβ in transformed leukocyte cell lines pointed to Purβ as the more distinguishing biomarker of myeloid cell differentiation status. Purβ ssDNA-binding activity was significantly increased in myeloid cells from AML patients but not from individuals with other myeloid-related diseases. The highest levels of Purβ activity were detected in myeloid cells from primary AML patients and from AML patients displaying other risk factors forecasting a poor prognosis. Collectively, these findings suggest that the enhanced ssDNA-binding activity of Purβ in transformed myeloid cells may serve as a unique and measurable phenotypic trait for improving prognostic risk stratification in AML. This article is protected by copyright. All rights reserved

Description: Branched-chain amino acid supplements consumed following exercise are widely used to increase muscle mass. Although both exercise (i.e., mechanical stimulation) and branched-chain amino acid leucine supplementation have been reported to stimulate muscle protein synthesis by activating the mammalian target of rapamycin (mTOR) signaling pathway independently, the mechanisms underlying their synergistic effects are largely unknown. Utilizing cultured differentiated C2C12 myotubes, we established a combination treatment model in which the cells were subjected to cyclic uniaxial mechanical stretching (4 h, 15%, 1 Hz) followed by stimulation with 2 mM leucine for 45 min. Phosphorylation of p70 S6 kinase (p70S6K), an mTOR-regulated marker of protein translation initiation, was significantly increased following mechanical stretching alone but returned to the baseline after 4 h. Leucine supplementation further increased p70S6K phosphorylation, with a greater increase observed in the stretched cells than in the non-stretched cells. Notably, the expression of L-type amino acid transporter 1 (LAT1), a stimulator of the mTOR pathway, was also increased by mechanical stretching, and siRNA-mediated knockdown partially attenuated leucine-induced p70S6K phosphorylation. These results suggest that mechanical stretching promotes LAT1 expression and, consequently, amino acid uptake, leading to enhanced leucine-induced activation of protein synthesis. LAT1 has been demonstrated to be a point of crosstalk between exercise- and nutrition-induced skeletal muscle growth. This article is protected by copyright. All rights reserved

Description: Fibroblast growth factor receptor (FGFR) 2 and its downstream signaling cascades, PI3K/AKT/mTOR is playing an important role in cell survival and proliferations. In this study, we firstly found that picrasidine Q (PQ), an alkaloid component extracted from Angelica keiskei species, has the capacity of anti-cell transformation and anti-cancer. After ligand shape similarity approach of PQ, we found that PQ targeted FGFR 2 and verified by FGFR2 kinase assay as well as computational docking model. FGFR2 highly expressed in esophageal cancer tissues and PQ inhibited fibroblast growth factor (FGF)-induced cell transformation. Furthermore, PQ inhibited cell proliferation and induced cell cycle arrest and apoptosis in KYSE30, KYSE410 and KYSE450 esophageal squamous cell carcinoma (ESCC) cells. It was confirmed by detecting of biological markers such as cyclinD1, cyclinD3 and cyclinB1 for cell cycle or cleaved caspase-7, caspase-3 and PARP for apoptosis. PQ targeting of FGFR2 kinase activities suppressed downstream target proteins including phosphorylation of AKT and mTOR but not MEK/ERK signaling pathways. Taken together, our results are the first to identify that PQ might be a chemopreventive and chemotherapeutic agent by direct targeting FGFR2 and inhibiting cell proliferation of ESCC cells. This article is protected by copyright. All rights reserved

Description: Coronary heart disease is a kind of disease which causes great injury to people world-widely. Although gene expression analyses had been performed previously, to our best knowledge, systemic co-expression analysis for this disease is still lacking to date. Microarray data of coronary heart disease was downloaded from NCBI with the accession number of GSE20681. Co-expression modules were constructed by WGCNA. Besides, the connectivity degree of eigengenes was analyzed. Furthermore, GO and KEGG enrichment analysis was performed on these eigengenes in these constructed modules. A total of 11 co-expression modules were constructed by the 3,000 up-regulated genes from the 99 samples with coronary heart disease. The average number of genes in these modules was 270. The interaction analysis indicated the relative independence of gene expression in these modules. The functional enrichment analysis showed that there was a significant difference in the enriched terms and degree among these 11 modules. The results showed that module 9 and module 10 played critical roles in the occurrence of coronary disease. Pathways of hsa00190(Oxidative phosphorylation)and (hsa01130: Biosynthesis of antibiotics) were thought to be closely related to the occurrence and development of coronary heart disease. Our result demonstrated that module 9 and module 10 were the most critical modules in the occurrence of coronary heart disease. Pathways as hsa00190(Oxidative phosphorylation) and (hsa01130: Biosynthesis of antibiotics) had the potential to serve as the prognostic and predictive marker of coronary heart disease. This article is protected by copyright. All rights reserved

Description: MicroRNA (miRNA) dysregulation has been associated with carcinogenesis in many cancers, including human colorectal cancer (hCRC). However, the effect and mechanism of miR-377-3p on CRC remains elusive. Herein, we first found that miR-377-3p was upregulated in CRC tissues and promoted tumorigenic activity by accelerating the G 1 -S phase transition, promoting cell proliferation and epithelial-mesenchymal transition (EMT) while repressing apoptosis in CRC cells. Glycogen synthase kinase-3β (GSK-3β) was a direct target of miR-377-3p, and upregulated by miR-377-3p. Knockdown of GSK-3β partly rescued miR-377-3p-mediated malignancy characteristics. Most importantly, we showed that miR-377-3p promoted carcinogenesis by activating NF-κB pathway. Taken together, our results first reported that miR-377-3p functions as an oncogene and promotes carcinogenesis via upregulating GSK-3β expression and activating NF-κB pathway in hCRC cells. This article is protected by copyright. All rights reserved

Description: Spinal cord injury (SCI) is a debilitating, costly, and common pathological condition that affects the function of central nervous system (CNS). To date, there are few promising therapeutic strategies available for SCI. To look for a suitable therapeutic strategy, we have developed a sublethal hypoxic preconditioning procedure using Fluorescence-activated cell sorting (FACS) analysis, LDH releasing and cell viability assays in vitro. Meanwhile, we have examined the benefits of neural stem cells (NSCs) transplantation prior to hypoxic preconditioning on functional recovery and potential mechanism via MRI screening, H&E and Nissl staining, immunofluorescence staining and Elisa assays. Our data showed that transplantation of hypoxic prconditioned NSCs could enhance neuronal survival, especially 5-TH + and ChAT + neurons, in the injured spinal cord to reinforce functional benefits. The hypoxia exposure upregulated HIF-1α, neurotrophic and growth factors including neurotrophin-3 (NT-3), glial cell-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) in vitro and in vivo. Furthermore, functional recovery, including locomotor and hypersensitivities to mechanical and thermal stimulation assessed via behavioral and sensory tests, improved significantly in rats with engraftment of NSCs after hypoxia exposure from day 14 post-SCI, compared with the control and N-NSCs groups. In short, the approach employed in this study could result in functional recovery via upregulating neurotrophic and growth factors, which implies that hypoxic preconditioning strategy could serve as an effective and feasible strategy for cell-based therapy in the treatment of SCI in rats. This article is protected by copyright. All rights reserved

Description: This study aims to investigate the effects of glucose transport l (Glut1) gene on proliferation, differentiation and apoptosis of colorectal cancer (CRC) cells by regulating the TGF-β/PI3K-AKT-mTOR signaling pathway. Immunohistochemistry was conducted to detect the positive Glut1 expression. Normal human CRC epithelial cells (CCD-18Co) and CRC cell line HCT116 were grouped into the control, blank, negative control (NC), and shGlut1-1 groups. RT-qPCR and Western blotting were performed to detect the expressions of Glut1, TGF-β1, PI3K, AKT, PTEN, mTOR, Bcl-2 and Bax. Protein expression of phosphorylated-PI3K (p-PI3K), p-S473-AKT, p-S389-S6K1, p-T70-4EBP1, Cleaved caspase-3 and Cleaved-PARP were detected. MTT assay, flow cytometry and colony formation assay were performed in order to detect cell viability, cell cycle and apoptosis, respectively. The positive expression rate of Glut1 in CRC tissues was 75% ± 8%, while in the adjacent normal tissues it was 0%. In comparison to adjacent normal tissues, CRC tissues had increased Glut1, TGF-β1, PI3K, AKT, mTOR and Bcl-2 expressions, and p-PI3K, p-S473-AKT, p-S389-S6K1 and p-T70-4EBP1 expressions; and decreased PTEN, Bax, Cleaved caspase-3 and Cleaved-PARP expressions. On comparison with the blank and NC groups, cells in the shGlut1-1 group showed decreased Glut1, TGF-β1, PI3K, AKT, mTOR and Bcl-2 expressions, and p-PI3K, p-S473-AKT, p-S389-S6K1 and p-T70-4EBP1 expressions; and increased PTEN, Bax, Cleaved caspase-3 and Cleaved-PARP expressions, along with more arrested cells in C0/C1 phase than in S phase and slower cell growth. These results suggested that silencing the Glut1 gene inhibited proliferation and promoted apoptosis of CRC cells by inactivating TGF-β/PI3K-AKT-mTOR signaling pathway. This article is protected by copyright. All rights reserved

Description: Pancreatic cancer is one of the major human malignant tumors severely endangering human health and life with high mortality due to the concealment of early symptoms and lack of effective therapies during advanced stages. The identification of pancreatic cancer stem cell functions has been as important strategy for understanding of pancreatic cancer biology and novel drug and therapy development. In the present study, we successfully isolated the pancreatic sphere-forming cells from pancreatic cancer cell line PANC-1 by sphere-forming method and we found that the sphere-forming ability and the cell migration rate of pancreatic sphere-forming cells were significantly inhibited by 5,7-DMF treatment, which was supported by the corresponding changes of several EMT biomarkers after being treated with 5,7-DMF. Moreover, we revealed here that the inhibition of pancreatic sphere-forming cells was mediated by the expression of FoxM1 gene, and also the expression of SOX2 gene was regulated by FoxM1 in pancreatic sphere-forming cells and involved in the inhibitory role of 5,7-DMF. These results provided important basis for the application of 5,7-DMF as a novel drug candidate for the pancreatic cancer treatment. This article is protected by copyright. All rights reserved

Description: Abnormal gametogenesis and embryonic development may lead to poor health status of the offspring. The operations involved in the assisted reproductive technologies (ARTs) occur during the key stage of gametogenesis and early embryonic development. To assess the potential risk of abnormal lipid metabolism in the liver of adult ARTs offspring, two ARTs mice models derived from preimplantation genetic diagnosis (PGD group) and in vitro cultured embryos without biopsy (IVEM group) were constructed. And control mice were from in vivo naturally conceived (Normal group). The results showed that ARTs offspring had increased body weight and body fat content comparing to normal group. An increasing volume and amount of lipid droplets as well as lipid droplet fusion were found in the hepatocytes of ARTs mice, and a significantly increased liver TG content was also shown in the ARTs mice, which due to the increased TG synthesis and decreased TG transport in the liver. All the results indicated that the manipulations involved in ARTs might play an important role in the lipid accumulation of adult offspring. By analyzing the DNA methylation profiles of 7.5dpc embryos, we proposed that methylation deregulation of the genes related to liver development in ARTs embryos might contribute to the abnormal phenotype in the offspring. The study demonstrated that ARTs procedures have adverse effect on liver development which resulted in abnormal lipid metabolism and induced the potential high risk of fatty liver in adulthood. This article is protected by copyright. All rights reserved

Description: Numerous anti-cancer agents inhibit cell cycle progression via a p53-dependent mechanism; however, other genes such as the proto-oncogene c-Myc are promising targets for anticancer therapy. In the present study, we provide evidence that ascochlorin, an isoprenoid antibiotic, is a non-toxic anti-cancer agent that induces G1 cell cycle arrest and p21 WAF1/CIP1 expression by downregulating of c-Myc protein expression. Ascochlorin promoted the G1 arrest, upregulated p53 and p21 WAF1/CIP1 , and downregulated c-Myc in HCT116 cells. In p53-deficient cells, ascochlorin enhanced the expression of G1 arrest-related genes except p53. Small interfering RNA (siRNA) mediated c-Myc silencing indicated that the transcriptional repression of c-Myc was related to ascochlorin-mediated modulation of p21 WAF1/CIP1 expression. Ascochlorin suppressed the stabilization of the c-Myc protein by inhibiting ERK and P70S6K/4EBP1 phosphorylation, whereas it had no effect on c-Myc degradation mediated by PI3K/Akt/GSK3β. The ERK inhibitor PD98059 and siRNA-mediated ERK silencing induced G1 arrest and p21 WAF1/CIP1 expression by downregulating c-Myc in p53-deficient cells. These results indicated that ascochlorin-induced G1 arrest is associated with the repression of ERK phosphorylation and c-Myc expression. Thus, we reveal a role for ascochlorin in inhibiting tumor growth via G1 arrest, and identify a novel regulatory mechanism for ERK /c-Myc. This article is protected by copyright. All rights reserved

Description: ABSTRACT In this study, we examined expression of nestin in the spinal cord, lung, kidney, stomach, colon and intestine tissues at different stages of embryos in patients with placenta previa. Fetuses of 75 patients with placenta previa were assigned to case group and 80 fetuses from healthy pregnant women with normal placenta who voluntarily terminated pregnancy to control group. Clinical data of pregnant women were collected at the time of admission. Blood from elbow vein was collected to determine expression of serum nestin. Tissues from spinal cord, lung, kidney, stomach, colon and intestine in 3-7 months fetuses of the two groups were extracted. Expression of nestin in tissues was detected by immunohistochemistry, western blotting and RT-qPCR. The mRNA expression of nestin in the case group was increased. Nestin expression was correlated with the gestational age, age of foetus and type of placenta previa in patients with placenta previa. Positive nestin expression was detected in the spinal cord, lung, kidney, stomach, intestine and colon tissues in normal and placenta previa embryo at Stage I. The positive cell density and nestin expression decreased at Stage II, and further decreased at Stage III. The case group had higher nestin mRNA and protein levels throughout human fetal development. Findings of this study suggested that, nestin, as a specific marker of neural precursor cells, was expressed in various tissues of the embryo in patients with placenta previa and nestin expression was lower with increased maturation of the embryo. This article is protected by copyright. All rights reserved

Description: Exosomes are released by normal and tumour cells, including those involved in breast cancer, and provide a means of intercellular communications. Exosomes with diameters ranging between 30-150 nm are involved in transferring biological information, via various lipids, proteins, different forms of RNAs, and DNA from one cell to another, and this can result in reprogramming of recipient cell functions. These vesicles are present in all body fluids, e.g., blood plasma/serum, semen, saliva, cerebrospinal fluid, breast milk, and urine. It has been recently reported that these particles are involved in the development and progression of different tumor types, including breast cancer. Furthermore, it has been suggested that exosomes have the potential to be used as drug transporters, or as biomarkers. This review highlights the potential roles of exosomes in normal and breast cancer cells and their potential applications as biomarkers with special focus on their potential applications in treatment of breast cancer. This article is protected by copyright. All rights reserved

Description: In this study, it was aimed to determine the doses of 4-methylcatechol causing cell death in rat insulinoma β-cells (INS-1), to find out the type of cellular death at these doses, and to investigate the molecular mechanism of cellular death occurring. More necrotic cells were observed than apoptosis with the administration of 350, 400, and 450 μM 4-methylcatechol. Lactate dehydrogenase levels, reactive oxygen species, mitochondrial potential loss, ATP and GTP losses increased at these doses. The JNK and ERK cellular pathway were screened. We observed an increase in p-RAF1 activity, the active JNK amount, the total c-Jun amount, while a decrease in p-RAF1 expression, the total JNK amount, JNK expression, ATF2 expression, active ERK and its expression and Elk1 expression. It was concluded that cells perform necrotic death by the following options: i) phosphorylated RAF1 activates the JNK pathway with the activity of transcription factor c-Jun, ii) Hsp 70 and Hsp 90 do not show a change inside the cell, rendering the JNK pathway active. This article is protected by copyright. All rights reserved

Description: ABSTRACT Surface localized microbial enolases' binding with human plasminogen has been increasingly proven to have an important role in initial infection cycle of several human pathogens.Likewise, surface localized Mycobacterium tuberculosis ( Mtb ) enolase also binds to human plasminogen, and this interaction may entail crucial consequences for granuloma stability. The current study is the first attempt to explore the plasminogen interacting residues of enolase from Mtb . Beginning with the structural modeling of Mtb enolase, the binding pose of Mtb enolase and human plasminogen was predicted using protein-protein docking simulations. The binding pose revealed the interface region with interacting residues and molecular interactions. Next, the interacting residues were refined and ranked by using various criteria. Finally, the selected interacting residues were tested experimentally for their involvement in plasminogen binding. The two consecutive lysine residues, Lys-193 and Lys-194, turned out to be active residues for plasminogen binding. These residues when substituted for alanine along with the most active residue Lys-429 i.e ., the triple mutant (K193A + K194A + K429A) Mtb enolase, exhibited40% reduction in plasminogen binding. It is worth noting that Mtb enolase lost nearly half of the plasminogen binding activity with only three simultaneous substitutions, without any significant secondary structure perturbation. Further, the sequence comparison between Mtb and human enolase isoforms suggests the possibility of selective targeting of Mtb enolase to obstruct binding of human plasminogen. This article is protected by copyright. All rights reserved

Description: Fibroblast growth factors (FGFs) are essential in regulating the formation of spermatogonial stem cells (SSCs). Here, we explored the effect of FGF8 on chicken SSCs formation by knockdown or overexpression of FGF8 in chicken embryonic stem cells (ESCs) both in vitro and in vivo . Our results showed that knockdown of FGF8 could facilitate the differentiation of ESCs into SSCs, overexpression of FGF8 could promote PGCs self-renewal, inhibit SSCs formation. This study further revealed the positive correlation between the expression level of FGF8 and MAPK/ERK signal. In the absence of FGF8, the expression of downstream genes such as FGFR2, GRB2, RAS, BRAF, RAF1 and MEK2 was not maintained, while overexpressing FGF8 enhances them. Thus, our study demonstrated that FGF8 can regulate germ cell fate by modulating the dynamic equilibrium between differentiation and self-renewal, which provides a new idea for the study of germ cell regulatory network. This article is protected by copyright. All rights reserved

Description: Introduction: Urinary tract regeneration using tissue engineering is one of the most challenging issues in the field of reconstructive urology. Cells seeded on scaffold are exposed to urine immediately after the implantation. The outcome of urinary bladder regeneration is depended on the ability of these cells to survive, proliferate and regenerate. The aim of this study was to compare a sensitivity of three different cell lines to urine in vitro . Material and methods: Three different cell lines were isolated from porcine bladder (urothelial cells, UCs and smooth muscle cells, SMCs) and adipose tissue (adipose-derived stem cells, ADSCs). Cell viability (MTT assay), proliferation (real-time cell analysis using xCELLigence system) and apoptosis/necrosis (flow cytometry) were analyzed after exposition to urine. Results: ADSCs were the most sensitive to urine compared to two other tested cell lines. Among the bladder cell lines the urothelial cells were more resistant to urine than SMCs. Twenty four hour incubation of UCs, SMCs and ADSCs with urine lead to ∼40%, ∼70% and ∼90% reduction of their viability, respectively. The mechanism of urine mediated cytotoxicity differed depending on the tested cell type. Conclusion: Urothelial and smooth muscle cells seem to be more suitable for urinary bladder regeneration compared to mesenchymal stem cells, however these cells have limited application especially in the case of urinary bladder cancer.

Description: DNA hypermethylation and the silencing of tumor suppressor genes caused by DNA hypermethylation is considered as a molecular hallmark of many kinds of cancers. Procaine, a local anesthetic, has been shown as a potential DNA methylation inhibitor in some types of cancers. However, the influence of procaine on DNA methylation regulation as well as the biological function in gastric cancer is still unknown. We report here that procaine represses the DNA-methylation level and promotes the proliferation arrest and apoptosis of gastric cancer cells. Global DNA methylation measurement demonstrates that procaine significantly reduces the global DNA methylation level. Analyses of the DNMTs expression and activity show procaine represses the activity, but not the expression, of DNMT1/DNMT3A. Further evidence on specific genes shows that procaine reduces the DNA methylation level in the promoter regions of CDKN2A and RARβ genes through abrogating the binding of DNMT1/DNMT3A towards these regions. This repression would not be reversed by the overexpression of DNMT1/DNMT3A. Moreover, RT-qPCR and luciferase report assays demonstrate that procaine leads to the upregulation of CDKN2A and RARβ due to the activation of the promoter of these genes. In the end, we test the function of procaine towards gastric cancer cells and find that procaine has the growth inhibitory and apoptosis inducement effect towards gastric cancer cells. Collectively, our data not only uncovers the regulation mechanisms of procaine to DNA methylation but also suggests an anti-tumor potential of procaine specific to the gastric carcinoma and provides a new therapeutic strategy for gastric carcinoma. This article is protected by copyright. All rights reserved

Description: Despite its explosive applications in genome engineering, CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats) has been developed into a versatile tool beyond its well-known nuclease function. In this prospect article, we summarize a few exciting "off-label" applications of CRISPR including manipulating DNA sequences, visualizing chromosomal loci in living cells, and modulating transcription and chromatin structures. These novel applications will likely elevate CRISPR tools into yet another level of sophistication and diversity, leading to many more exciting cell biological discoveries. Since its inception as an exciting new genome-engineering platform, the CRISPR/Cas (CRISPR associated protein) system has evolved to be more than just a tool for targeted mutagenesis. Adapted from its natural role as part of the bacterial adaptive immune system, the CRISPR system has been simplified to consist of a single-guide RNA (sgRNA) and Cas nuclease (most commonly spCas9 derived from Streptococcus pyogenes ). Comprised of nuclease binding stem-loop structures and a ∼20 bp 5'- overhang for sequence-specific DNA recognition, a Cas9-loaded sgRNA scans the DNA for its complementary target sequence. Novel applications for the CRISPR/Cas system have emerged with the disabling of the nuclease domains of the Cas9 protein, resulting in a catalytically-inactive Cas9 nuclease (dCas9). This was done by introducing two point mutations, H840A to the HNH domain and D10A to the RUVC domain, which eliminated its ability to cleave either strand of DNA while maintaining its sequence-specific DNA recognition capability (Jinek et al., 2012). Acting as a programmable DNA binding protein, the CRISPR gene-editing system may now be repurposed to carry out new functions such as manipulating gene expression and labeling loci for dynamic cell imaging. In this prospect article, we discuss a few of the new and exciting uses of the CRISPR system, beyond its most popular application as a designer nuclease. This article is protected by copyright. All rights reserved

Description: Objective : To investigate the biocompatibility and differentiation of human brain-derived neurotrophic factor (hBDNF) gene-modified bone marrow mesenchymal stem cells (hBDNF-rMSCs) in a functionalized self-assembling peptide hydrogel. Methods : hBDNF were engineered in rMSCs using adenovirus vector and the enhanced green fluorescence protein (eGFP) was used as a reporter gene. MSC-specific surface markers (CD90,CD29 and CD45)were used for identifying rMSCs. Fluorescence microscope was used to detect the transfection of rMSCs. hBDNF-rMSCs and control cells (eGFP-rMSCs) were seeded in a functional self-assembling peptide hydrogel (RADA16-PRG hydrogel) and a control hydrogel (RADA16 hydrogel). Cells were divided into three groups (hBDNF-rMSCs + RADA16 Hydrogel, hBDNF-rMSCs + RADA16-PRG Hydrogel and eGFP-rMSCs+ RADA16-PRG Hydrogel) and a control group (eGFP-rMSCs + RADA16 Hydrogel). Cell growth, cell proliferation, expression of hBDNF-mRNA, the level of hBDNF, neuron-specific enolase (NSE), and glial fibrillary acidic protein (GFAP) protein were analyzed for each group. Result : rMSCs were positive for CD90 and CD29 and negative for CD45, green fluorescence was strongly visible at 72h after transfection. Compared with control group,the expression of hBDNF-mRNA and levels of hBDNF protein in both hBDNF group were significantly increased ( P  〈 0.01),the cell growth, cell proliferation and levels of NSE and GFAP protein were significantly increased in three groups ( P  〈 0.01). Cell growth, cell proliferation,expression of hBDNF-mRNA and levels of hBDNF,NSE and GFAP protein in hBDNF-rMSCs + RADA16-PRG Hydrogel Group were significantly higher than that of hBDNF-rMSCs + RADA16 Hydrogel Group ( P  〈 0.01). Conclusion : Bone marrow mesenchymal stem cells can be induced into neural cells by the human brain-derived neurotrophic factor gene in a RADA16-PRG functionalized self-assembling peptide hydrogel. This article is protected by copyright. All rights reserved

Description: The study aimed to investigate the mechanism by which the sonic Hedgehog (SHH) gene silencing acts upon epithelial-mesenchymal transition (EMT), proliferation, invasion and migration of cervical cancer (CC) cells via the Hedgehog signaling pathway. RT-qPCR and western blotting were all employed to detect the SHH mRNA and protein expressions. HeLa and CasKi cells were cultured and subsequently divided into the blank, negative control (NC) and SHH-RNAi groups. A cell counting kit-8 (CCK-8) assay was utilized for cell proliferation. Cell migration and invasion ability were evaluated through scratching test and Transwell assay. The mRNA and protein expressions of the Hedgehog signaling pathway-related factors were detected using RT-qPCR and western blotting, respectively. After tumor xenograft in nude mice, tumor growth was subsequently observed. SHH mRNA and protein expressions were greater in the SHH-RNAi group than in the blank and NC groups. Compared with the blank group and NC groups, the SHH-RNAi group displayed inhibited levels of proliferation, migration, invasion abilities, as well as a decreased in the Hh signaling pathway-related factors, as well as a reduction in the mRNA and protein expressions of N-cadherin and Vimentin, however on the contrary increased expressions of E-cadherin were observed. Following tumor xenograft in nude mice, tumor growth was exhibited vast levels of inhibition, particularly in the SHH-RNAi group in comparison to the blank and the NC groups. During the study it was well established that SHH gene silencing suppresses EMT, proliferation, invasion and migration of CC cells through the repression of the Hedgehog signaling pathway. This article is protected by copyright. All rights reserved

Description: Bleomycin mediated inflammatory pathway is known to play an important role in the up regulation of oxidative stress. IL-17A is a pro-inflammatory cytokine involved in the modulation of fibrosis. The complex underlying mechanism for the said phenomenon remains unclear. This newly defined investigation was designed to understand the changes associated with 1L-17A mediated up-regulation of p53 and PAI-1 expression and the role of curcumin in attenuating this process. A549 cells were treated with bleomycin (BLM) and IL-17A to induce the inflammatory response in vitro . Curcumin, a known anti-inflammatory bioactive compound was administered as an intervention. Cytotoxicity in the treatment groups was assessed using Methyl thiazolyl tetrazolium (MTT) assay. Cell migration was evaluated using scratch assay. Protein expressions were studied using western blot analysis for the downstream effector molecules of IL-17A mediated inflammatory pathways. In MTT assay, BLM treatment showed cytotoxicty upto 88% at a concentration of 1000 µM after 48 h of treatment. Cell migration assay results revealed that curcumin blocked the migration of cells to the area of the scratch. BLM treatment to the cells significantly induced the expression of pro-inflammatory cytokine IL-17A, which in turn modulated p53-PAI-1 expression. Bioactive compound curcumin showed anti-inflammatory and anti-apoptotic activity. Curcumin also regulated the BLM and IL-17A mediated changes in p53–PAI-1 expression. Curcumin has the ability to regulate inflammatory cytokines during BLM induced injury and their effect on p53-PAI-1 expression. It can be used as a potential anti-inflammatory and anti-fibrinolytic component for intervening the epithelial cell damage. This article is protected by copyright. All rights reserved

Description: ABSTRACT Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are clinical emergencies with no effective pharmaceutical treatment. This study aims to determine the protective effects of Nrf2-transfected human amniotic mesenchymal stem cells (hAMSCs) against lipopolysaccharide (LPS)-induced lung injury in mice. hAMSCs stably transfected with Nrf2 or green fluorescent protein control were transplanted into male C57BL/6 mice via the tail vein 4 h after intratracheal instillation of LPS. At 3, 7 and 14 days after cell transplantation, total lung injury score (the Smith score) was determined by hematoxylin and eosin staining. Lung fibrosis was assessed by Masson's trichrome staining. Alveolar epithelial apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nick end labeling staining. The plasma levels of interleukin (IL)-1β, IL-6, and IL-10 were determined by enzyme-linked immunosorbent assays (ELISA). The homing and differentiation of hAMSCs into type II alveolar epithelial (AT II) cells were examined by immunofluorescent staining and/or western blot analysis. Nrf2 mRNA and protein expression in lungs were examined by qRT-PCR and western blot analysis, and DNA-binding activity of Nrf2 was detected by ELISA. We found that, compared with control hAMSCs, treatment with Nrf2-overexpressing hAMSCs led to further reduced lung injury, lung fibrosis, and inflammation in LPS-challenged mice. Nrf2-overexpressing hAMSCs also exhibited increased cell retention in the lung, more efficient differentiation into AT II cells, and more prominent effects on the increased mRNA and protein expression as well as DNA-binding activity of Nrf2 than control. These results support Nrf2-overexpressing hAMSCs as a potential cell-based therapy for clinical ALI/ARDS. This article is protected by copyright. All rights reserved

Description: This study aimed to clone the Stearoyl-CoA desaturase 1 (SCD1) gene derived from Xuhuai goat ( Capra hircus ), and analyze the sub-cellular localization in cells and tissues. The cDNA was cloned by reverse transcription polymerase chain reaction (RT-PCR). pEGFP-SCD1 vector was constructed to detect sub-cellular localization and tissue distribution. pEGFP-SCD1 was transfected into NIH-3T3 cells using polyethylene imine (PEI) and observed under fluorescence inversion microscope system 48 h after transfection. The expression level of SCD1 was detected by RT-PCR. Testicular injection was used to produce transgenic mice with goat SCD1 gene. DNA and protein were extracted from the tail tissue of F 1 mice. The expression of exogenous gene in the F 1 generation was detected in both DNA and protein. The results showed that the coding sequence (CDS) fragments of Capra hircus SCD1 gene was 1074 bp and encodes 360 amino acids. RT-PCR results showed that SCD1 could be expressed successfully in NIH-3T3 cells in vitro . Sub-cellular localization analysis showed that pEGFP-SCD1 fusion protein located in the cytoplasm. It can be concluded that transgenic mice with goat SCD1 expressed in sperm and tail tissue was successfully produced in the F 1 mice generation. This article is protected by copyright. All rights reserved

Description: This study aims to explore the correlation between interleukin-1 (IL-1) and epilepsy in rats when treated with low-frequency electrical stimulation via the RhoA/ROCK signaling pathway. Twenty-four SD rats were elected for this study, among which six rats were assigned as the normal group. And 16 rat models with epilepsy were successfully established and assigned into the model group, the ES group and the ES + IL-8 group, with each group comprising of six rats. The seizure frequency and duration was recorded. Electroencephalogram (EEG) power was detected at α1, α2, β, θ and δ. The mRNA expressions of IL-1β and IL-1R1 were detected using reverse transcription quantitative polymerase chain reaction (RT-qPCR), and the protein expressions of RhoA, ROCK I and ROCK II were detected by western blotting. In comparison with the model group, the seizure frequency duration, the power of δ, θ, α1, α2 and β, the mRNA and protein expressions of IL-1β and IL-1R1, the expressions of RhoA and ROCK I proteins, and the ratio of RhoA protein between membrane and cytosol decreased in the ES group, while the expression of ROCK II increased (all P 〈 0.05). There was no significant difference in these indexes between the ES + IL-8 group and the model group (all P 〉 0.05). These findings signified that IL-1 might inhibit the efficacy of low-frequency ES for epilepsy via the RhoA/ROCK signaling pathway, which may provide a theoretical basis for clinical treatment of epilepsy. This article is protected by copyright. All rights reserved

Description: Autologous chondrocyte implantation (ACI) has emerged as a new approach to cartilage repair through the use of harvested chondrocytes. But the expansion of the chondrocytes from the donor tissue in vitro is restricted by limited cell numbers and dedifferentiation of chondrocytes. In this study, we used four types of hydrogels including agarose, alginate, Matrigel and collagen type I hydrogels to serve as cell substrates and investigated the effect on proliferation and phenotype maintenance of chondrocytes. As a substrate for monolayer culture, collagen facilitated cell expansion and effectively suppressed the dedifferentiation of chondrocytes, as evidenced by fluorescein diacetate / propidium iodide (FDA/PI), hematoxylin-eosin staining (HE), Safranin O, immunofluorescenceassay, biochemistry analysis and quantitative real-time polymerase chain reaction (qRT-PCR). Compared with that in agarose gels, alginate and Matrigel, collagen accelerated cell proliferation and enhanced the expression of cartilage specific genes such as ACAN, SOX9 and COLII more markedly. Furthermore, significantly lower expression of COL I (an indicator of dedifferentiation) and COL X (the chondrocyte hypertrophy marker) was present in collagen group than in other groups. This indicated that collagen substrate can better support chondrocyte growth and maintain cell phenotype, due to that it might serve as a cartilage-like ECM to provide adhesive site for chondrocytes. In summary, collagen hydrogel is a promising cell substrate for chondrocytes culture for ACI. This article is protected by copyright. All rights reserved

Description: β-thalassemia is a common autosomal recessive disorder characterized by a deficiency in the synthesis of β-chains.Evidences show that increased HbF levels improve the symptoms in patients with β-thalassemia or sickle cell anemia. In this study, ZFN technology was applied to induce a mutation in the binding domain region of SOX6 to reactivate γ-globin expression. The sequences coding for ZFP arrays were designed and sub cloned in TDH plus as a transfer vector. The ZFN expression was confirmed using Western blot analysis. In the next step, using the site-directed mutagenesis strategy through the overlap PCR,a missense mutation (D64V) was induced in the catalytic domain of the integrase gene in the packaging plasmid and verified using DNA sequencing.Then, the integrase minus lentivirus containing ZFN cassette was packaged. Transduction of K562 cells with this virus was performed.Mutation detection assay was performed. The indel percentage of the cells transducted with lenti virus containing ZFN was 31%. After 5 days of erythroid differentiation with 15µg/ml cisplatin, the levels of γ–globin mRNA were 6-fold in the cells treated with ZFN compared to untreated cells. In the meantime, the measurement of HbF expression levels was carried out using hemoglobin electrophoresis and showed the same results. Integrase minus lentivirus can provide a useful tool for efficient transient gene expression and helps avoid disadvantages of gene targeting using the native virus.The ZFN strategy applied here to induce indel on SOX6 gene in adult erythroid progenitors may provide a method to activate fetal hemoglobin expression in individuals with β-thalassemia. This article is protected by copyright. All rights reserved

Description: In this study, we aim to determine the function of miR-124 on gastric cancer (GC) cells and the underlying mechanism that involves jaddeg1 (JAG1) and the Notch signaling pathway. GC tissues and adjacent tissues from 100 patients suffering from GC were selected. GC SGC-7901 and AGS cells were selected and grouped into control, mimic-NC, miR-124 mimic, inhibitor-NC, miR-124 inhibitor, and miR-124 inhibitor + si-JAG1 groups. RT-qPCR and a western blotting assay were conducted to detect the expression of miR-124, JAG1 and Notch signaling pathway-related proteins (NICD, HES1 and HES5). MTS, wound-healing, transwell assay and flow cytometry were performed to detect cell proliferation, migration, invasion, cell cycle distribution and apoptosis, respectively. Compared with adjacent tissues, a lower miR-124 expression and higher JAG1 expression were found in GC tissues. JAG1 is a direct target gene of miR-124. Compared with the control group, the expression of JAG1, NICD, HES1, and HES5, cell invasion, migration and proliferation in the miR-124 mimic group were decreased, while the apoptosis rate was increased and cells were arrested at the G0/G1 phase. Compared with the miR-124 inhibitor group, the expression of JAG1, NICD, HES1, and HES5, cell invasion, migration and proliferation in the miR-124 inhibitor + si-JAG1 group were decreased, while the apoptosis rate and cell ratio at the G0/G1 phase were increased. The demonstration that miR-124 inhibits GC cell growth supports the concept that miR-124 functions as a tumor suppressor by a mechanism that involves translational repression of the JAG1 and the inhibition of Notch signaling pathway. This article is protected by copyright. All rights reserved

Description: P73 antisense RNA 1T (TP73-AS1 or PDAM) is a long non-coding RNA, which can regulate apoptosis through regulation of p53 signaling-related anti-apoptotic genes. An abnormal change of TP73-AS1 expression was noticed in cancers. The effects of TP73-AS1 in breast cancer (BC) growth and the underlying mechanism remain unclear so far. In the present study, the effect of TP73-AS1 in BC cell lines and clinical tumor samples was detected so as to reveal its role and function. In the present study, TP73-AS1 was specifically upregulated in BC tissues and BC cell lines and was correlated to a poorer prognosis in patients with BC. TP73-AS1 knocking down suppressed human BC cell proliferation in vitro through regulation of TFAM. In our previous study, we demonstrated that miR-200a inhibits BC cell proliferation through targeting TFAM; here we revealed that TP73-AS1 could regulate miR-200a through direct targeting. Moreover, TP73-AS1 might compete with TFAM for miR-200a binding thus to promote TFAM expression. Data from the present study revealed that TP73-AS1 promoted BC cell proliferation through acting as a competing endogenous RNA (ceRNA) by sponging miR-200a. In conclusion, we regarded TP73-AS1 as an oncogenic lncRNA promoting BC cell proliferation and a potential target for human BC treatment. This article is protected by copyright. All rights reserved

Description: ABSTRACT Nucleotide-binding oligomerization domain containing 2 (NOD2)-induced signal transduction and cytokine production is regulated by a number of factors. However, the feedback effect of the pro-inflammatory TNF-α on NOD2-induced inflammation is not fully understood. In this study, we found unexpectedly that TNF-α up-regulated NOD2 ligand MDP-induced production of the CXC chemokines, including CXCL1, 2 and 8, and the pro-inflammatory cytokines, including IL-1β, IL-6 and TNF-α, in a dose-dependent manner at both mRNA and protein levels in monocytic THP-1 cells. Though TNF-α induced the up-regulation of ubiquitin-editing enzyme A20, an important negative regulator for Toll-like receptor- and NOD2-induced inflammatory responses, the over-expression of A20 by gene transfer did not reversed MDP-induced production of cytokines, suggested that A20 did not regulate the functions of NOD2 in THP-1 cells. Meanwhile, we found that TNF-α up-regulated NOD2 and its down-stream adaptor protein RIP2 at both mRNA and protein levels. MDP induced the activation of ERK, JNK, p38 and NF-κB, and TNF-α pre-treatment augmented this activation. The results from pharmacological inhibition assay showed that cytokine production was dependent on MAPK signaling. In addition, we found that the pre-treatment of THP-1 cells with MDP down-regulated the mRNA levels of cytokine induced by MDP re-treatment. MDP pre-treatment up-regulated NOD2, but down-regulated RIP2, and down-regulated NOD2 signal transduction induced by MDP re-stimulation. Taking together, these results suggested that TNF-α is a positive regulator for NOD2 functions via up-regulation of NOD2 and its signal adaptor RIP2, and TNF-α-induced A20 does not regulate MDP-induced inflammatory responses in THP-1 cells. This article is protected by copyright. All rights reserved

Description: The targeting protein for Xenopus kinesin-like protein 2 (TPX2) is associated with the metastasis and prognosis of bladder cancer. p53 is closely related to the progression of bladder cancer. Human glioma pathogenesis-related protein 1 (GLIPR1) is a p53 target gene with antitumor activity. This study aims to explore the interplay between TPX2, p53 and GLIPR1 and its correlation with cell proliferation, invasion and tumor growth in bladder cancer. Here, Western blot and qRT-PCR analysis revealed that TPX2 at both mRNA and protein levels was up-regulated in bladder carcinoma tissues compared to their paired adjacent normal tissues. Additionally, tissues expressing high TPX2 level exhibited high p53 level and low GLIPR1 level. The expressions of TPX2 and p53 in non-muscle-invasive bladder cancer cells (KK47 and RT4) were lower than those in muscle-invasive bladder cancer cells (T24, 5637, and UM-UC-3), while GLIPR1 showed the converse expression pattern. Further investigation revealed that TPX2 activated the synthesis of p53; and GLIPR1 is up-regulated by wild-type (wt)-p53 but not affected by mutated p53; Additionally, GLIPR1 inhibited TPX2. These data suggested a TPX2-p53-GLIPR1 regulatory circuitry. Meanwhile, TPX2 overexpression promoted while overexpression of GLIPR1 or p53 inhibited bladder cancer growth. Interestingly, in T24 cells with mutated p53, p53 silence suppressed bladder cancer growth. This study identified a novel TPX2-p53-GLIPR1 regulatory circuitry which modulated cell proliferation, migration, invasion, and tumorigenicity of bladder cancer. Our findings provide new insight into underlying mechanisms of tumorigenesis and novel therapeutic options in bladder cancer. This article is protected by copyright. All rights reserved

Description: Previously, we synthesized a non-viral vector containing disulfide bond by polymerization of agamatine (AGM) and N, N'-cystaminebisacrylamide (CBA). In this study, we investigated the transfection efficiency of disulfide bond (-SS-) containing AGM-CBA polymer in gene delivery into NIH/3T3 cells, and examined the factors affecting its transfection efficiency by comparing with polyethylenimine (PEI). In addition, experiments were carried out to determine the mechanisms of cell entry pathways and intracellular behavior of AGM-CBA/pDNA polyplexes. The transfection efficiency of AGM-CBA/pDNA with different weight ratios and different amounts of pDNA were measured and the pathways mediated transfection processes were studied by using various endocytosis inhibitors. To determine the intracellular behavior of AGM-CBA/pDNA polyplexes, the transfection efficiencies of AGM-CBA/pDNA and PEI/pDNA polyplexes with different combination structures were determined by using reporter gene and fake plasmid DNA. The transfection efficiency of AGM-CBA/pDNA polyplexes was correlated with its weight ratio of AGM-CBA and pDNA, and the amount of pDNA. Both AGM-CBA/pDNA and PEI/pDNA polyplexes entry into cell by clathrin and caveolae mediated endocytic pathways. However, AGM-CBA/pDNA showed different intracellular behavior in NIH/3T3 cells compared to PEI/pDNA polyplexes. It was hypothesized that disulfide bond in AGM-CBA could be an important factor contributing to its intracellular behavior and better transfection efficiency. Overall, AGM-CBA demonstrated better transfection efficiency and lower cytotoxicity than PEI in NIH/3T3 cells as a gene delivery vector. This article is protected by copyright. All rights reserved

Description: ABSTRACT This study aimed to explore effects of CNP on ventricular remodeling following myocardial ischemia-reperfusion (I/R) injury through the NPRB/cGMP signaling pathway. Rat cardiomyocyteswere assigned into: control, I/R, I/R + CNP, and I/R+ 8-Br-cGMP groups. ELISA, qRT-PCR and western blotting were used to detect cGMP content and expression, respectively. After model establishment of I/R rats, normal control, CNP -/- control, I/R, and CNP -/- groups were set. Indexes of heart were detected using echocardiography and hemodynamics. ELISA was used to measure serum CNP, cGMP, LDH, cTn I, CK-MB, TNF-α, and IL-6 levels. Myocardial infarct was identified by TTC staining, and apoptosis conditions by TUNEL staining. QRT-PCR and western blotting were adopted to detect expressions of CNP, NPRB, cGMP, and apoptosis-related genes. Compared with control group, cGMP contents and expression in the I/R, I/R + CNP and I/R + 8-Br-cGMP groups were decreased. Levels of LVEDV, LVESV, LVDS, LVDD, IVSD, LVM, LVEDP, and LVSP were higher in the I/R, CNP -/- control and CNP -/- groups than normal control group while LVEF, SV, CO, and ± dp/dtmax were lower. Compared with the normal control group, LDH, cTn I, CK-MB, TNF-α, and IL-6 were higher in the I/R, CNP -/- control and CNP -/- groups; pathological changes and myocardial infarction were observed in the I/R, CNP -/- control, and CNP -/- groups; expressions of apoptosis-related genes in those groups were higher; while CNP, NPRB, cGMP and Bcl-2 expressions were decreased. We came to the conclusion that gene knockdown of CNP blocks the NPRB/cGMP signaling pathway, thereby aggravating myocardial I/R injury and causing ventricular remodeling in rats. This article is protected by copyright. All rights reserved

Description: ABSTRACT Hypoxia is an element intrinsic to most solid-tumor microenvironments, including that of osteosarcoma (OS), and is associated with resistance to therapy, poor survival, and a malignant phenotype. The purpose of the present study was to investigate the role and underlying mechanism of SUMO-specific protease 1 (SENP1)/hypoxia-inducible factor-1α (HIF-1α) feedback loop in hypoxic microenvironment of OS. We observed that the expression of SENP1 was remarkably upregulated in OS cells. Additionally, there was a concomitant high expression of HIF-1α and SENP1 in MG-63 cells under a hypoxic microenvironment. Interestingly, blockage of HIF-1α repressed the enhancement of SENP1. Moreover, knockdown of SENP1 with siRNA notably inhibited cell viability and accelerated cell apoptosis accompanied by a decrease in the expression of Bcl-2 and an increase in the expression of Bax in MG-63 cells following exposure to hypoxia. Furthermore, repression of SENP1 dramatically suppressed cell invasive ability through modulating epithelial-mesenchymal transition (EMT) marked genes as reflected by the upregulation of E-cadherin, as well as the downregulation of vimentin and N-cadherin under hypoxic conditions. Most importantly, SENP1 positively regulated HIF-1α expression level in the setting of hypoxic; subsequently, depletion of SENP1 expression markedly ameliorated vascular endothelial growth factor (VEGF) production triggered by hypoxia. Taken together, positive feedback loop between HIF-1α and SENP1 in the regulating of the process of cell proliferation, invasion and EMT in OS cells under hypoxic conditions, suggesting that the SENP1/HIF-1α axis may serve as a new potential therapeutic agent for OS treatments. This article is protected by copyright. All rights reserved

Description: Renilla Luciferase (RLuc) is a blue light emitter protein which can be applied as a valuable tool in medical diagnosis. But due to lack of the crystal structure of RLuc-ligand complex, the functional motions and catalytic mechanism of this enzyme remain largely unknown. In the present study, the active site properties and the ligand-receptor interactions of the native RLuc and its red-shifted light emitting variant (Super RLuc 8) were investigated using molecular docking approach, molecular dynamics (MD) analysis, and MM-PBSA method. The detailed analysis of the main clusters led to identifying a lid-like structure and its functional motions. Furthermore, an induced-fit mechanism is proposed where ligand-binding induces conformational changes of the active site. Our findings give an insight into the deeper understanding of RLuc conformational changes during binding steps and ligand-receptor pattern. Moreover, our work broaden our understanding of how active site geometry is adjusted to support the catalytic activity and red-shifted light emission in Super RLuc 8. This article is protected by copyright. All rights reserved

Description: ABSTRACT It has recently been shown that miR-622 plays a tumor suppressive role in many human cancers. However, the exact function and underlying mechanism are still unknown. Here, we reported that the level of miR-622 is clearly reduced in human glioma tissues in comparison with normal brain tissues and is negatively correlated with the histological grades. Additionally, ectopically expressed miR-622 significantly inhibited cell proliferation and induced cell cycle arrest at the G0/G1 phase in glioma cells. Furthermore, the bioinformatics analysis revealed that YAP1 possesses putative miR-622 -binding sites within its 3'UTR. Consequently, an elevated miR-622 level was found to suppress the luciferase reporter activity of YAP1 3'UTR, and the effect was diminished by the deletion of the miR-622 seed binding site. In addition, the level of YAP1 protein expression was significantly decreased after the overexpression of miR-622. These results indicate a negative link between miR-622 and YAP1 and further confirm that YAP1 is a direct target of miR-622, suggesting that miR-622 could be a new important therapeutic strategy for gliomas treatment. This article is protected by copyright. All rights reserved

Description: ABSTRACT Peptic ulcer disease is one of the major challenges in public health globally and new evidence shows that it can be controlled by targeting the histamine H 2 receptor (H 2 R). Recently, a number of H 2 R antagonists have been synthesized and used to block the action of histamine on the parietal cells in the stomach and decrease the acid production. In this study, we modeled the H 2 R by homology modeling using the 3-D crystal structure and this model was validated based on free energy and amino acid residues present in the allowed regions of a Ramachandran plot. We used this 3-D model for screening of highly potent drugs using molecular docking. We found cimetidine, cimetex, and famotidine as the most potent drugs based on the binding affinity of drug-protein interactions. We also generated a cellular network for H 2 R that could be useful for better understanding of cellular mechanism and drug targets. These findings provide a new insight into the development of suitable, specific and effective anti-ulcer drugs for a most effective treatment of ulcerous diseases. This article is protected by copyright. All rights reserved

Description: The symptoms of ovarian cancer at early stages are usually absent which makes the diagnosis in its early stages exceedingly difficult. Previous research has proven that ovarian cancer is a genetic disease, which depends on the alteration of multi-cancer related genes and anti-cancer genes, multi-stages and multi-pathways, involving a variety of oncogene activation and anti-oncogene inactivation. For a better understanding of the prognostic classification of ovarian cancer, gene expression profiles were used to analyse the prognostic factors of ovarian cancer, and the prognostic model was used to classify the ovarian cancer samples. The ovarian cancer samples data were downloaded from TCGA dataset. Rebust likelihood-based survival model was built to find the key genes that could function as prognostic markers. The samples were classified by unsupervised hierarchical clustering. Furthermore, Kaplan Meier survival analysis was used to analyze the differences in the prognosis of the samples. The prognostic model was used to classify the samples, and then the best classification model was selected as the prognostic model of ovarian cancer. Finally, GEO datasets were used for external data validation. A total of 886 genes with influence on prognosis was obtained. Then genomic combinations of 11 genes were screened out by random sampling. Then the active number of influential factors was counted based on the expression level of featured genes. When the number of influencing factors is ≥ 7, the prognosis difference among these genes is the largest (p-value = 0.000775); and this was chosen as the final Classification model. To summary, a prognostic 11genes expression model was preliminarily built to classify the ovarian cancer samples. This article is protected by copyright. All rights reserved

Description: The development of new diabetes drugs continues to be explored. Loureirin B, a flavonoid, extracted from Dracaena cochinchinensis , has been confirmed to increase insulin secretion and decrease blood glucose levels. For searching the promotion of insulin secretion with the treatment of loureirin B, experiments were employed based on cell experiments and computational methods. Firstly, promotion of insulin secretion was dependent on extracellular glucose concentration. At the genetic level, loureirin B enhanced the relative mRNA level of Pdx-1 and MafA . Meanwhile the intracellular level of ATP increased due to the continuous absorption of glucose. Further experiments showed that the currents of K ATP channel on Ins-1 cells were inhibited and the voltage-dependent calcium channels were subsequently activated. The increase of Cx43 protein expression might mediate the Ca 2+ to the intracellular. Through computational simulation, we hypothesized that loureirin B might interact with K ATP channels to promote insulin secretion. In conclusion, it could be concluded that loureirin B promoted insulin secretion mainly through increasing mRNA level of Pdx-1 , MafA , intracellular ATP level, inhibiting the K ATP current, influx of Ca 2+ to the intracellular. This article is protected by copyright. All rights reserved

Description: ABSTRACT Genistein has been reported to exert beneficial effects on type 2 diabetes mellitus (T2DM); however, the underlying molecular mechanisms involved therein have not been clearly elucidated. To address this question, the effect of the genistein on the expression of phosphoenolpyruvate carboxykinase (PEPCK), and glucose production in HepG2 cells and in alloxan-induced diabetic mice was investigated. HepG2 cells were exposed to diferrent concentration of genistein in presence or absence of modulators, and the expression of cytosolic PEPCK (PEPCK-C) and the signalling pathways was studied. Further, the biological relevance of the in vitro study was tested in alloxan induced diabetic mice. Genistein lowered PEPCK-C expression and glucose production in HepG2 cells accompanied with increased in phosphorylation states of AMPK, MEK½, ERK½, and CRTC2. Treatment with the AMPK inhibitor (compound C) enhanced genistein-induced MEK½ and ERK½ activity indicating a potential cross-talk between the two signalling pathways. In vivo , genistein also reduced fasting glucose levels accompanied with reduced PEPCK-C expression and increased in AMPK and ERK½ phosphorylation states in the liver of genistein-treated alloxan-induced diabetic mice. Genistein fulfills the criteria of a suitable anti-diabetic agent by reducing glucose production and inhibiting PEPCK-C expression in HepG2 cells and also in alloxan-induced diabetic mice. These results indicate that genistein is an effective candidate for preventing T2DM through the modulation of AMPK-CRTC2 and MEK/ERK signalling pathways, which may allow a novel approach to modulate dysfunction in hepatic gluconeogenesis in T2DM. This article is protected by copyright. All rights reserved

Description: ABSTRACT Human dental pulp exposed to hypoxic conditions induces cell death accompanied by autophagy. However, the role of hypoxia-induced autophagy in human dental pulp cells (HDPCs) is unclear. The present study aimed to investigate the role of autophagy in hypoxia-induced apoptosis of HDPCs. Cobalt chloride (CoCl 2 ) treated HDPCs, to mimic hypoxic conditions, decreased cell viability. Also, apoptosis-related signal molecules, cleaved caspase-3 and PARP levels, were enhanced in CoCl 2 -treated HDPCs. HDPCs exposed to CoCl 2 also promoted autophagy, showing upregulated p62 and microtubule-associated protein 1 light chain 3 (LC3)-II levels, typical autophagic markers, and increased acidic autophagolysosomal vacuoles. Autophagy inhibition by 3 methyladenine (3MA) or RNA interference of LC3B resulted in increased levels of cleaved PARP and caspase-3, and the release of cytochrome c from mitochondria into cytosol in the CoCl 2 -treated HDPCs. However, autophagy activation by rapamycin enhanced the p62 and LC3-II levels, whereas it reduced PARP and caspase-3 cleavage induced by CoCl 2. These results revealed that CoCl 2 -activated autophagy showed survival effects against CoCl 2 -induced apoptosis in the HDPCs. CoCl 2 upregulated HIF-1α and decreased the phosphorylation of mTOR/p70S6K. HIF-1α inhibitor, YC-1 decreased p62 and LC3-II levels, whereas it augmented PARP and caspase-3 cleavage in response to CoCl 2 . Also, YC-1 enhanced the phosphorylation of mTOR and p70S6K suppressed by CoCl 2 , demonstrating that CoCl 2 -induced autophagy via mTOR/p70S6K is mediated by HIF-1α. Taken together, these finding suggest that CoCl 2 -induced autophagy mediated by the mTOR/p70S6K pathway plays a protective role against hypoxic stress in HDPCs. This article is protected by copyright. All rights reserved

Description: ABSTRACT The aim of this study was to investigate the correlation of expression of IGF1R-RACK1-STAT3 and Bcl-xl in nasopharyngeal carcinoma (NPC) with the clinicopathological features and the prognosis of NPC. Our study selected 215 NPC tissues and 178 chronic nasopharyngitis tissues (control group). Positive expression rates of IGF1R, RACK1, STAT3 and Bcl-xl were tested by immunohistochemical method, and expression of IGF1R, RACK1, STAT3, Bcl-xl, Bcl-2 and Bax by western blotting. Correlation of IGF1R, RACK1, STAT3 and Bcl-xl with the clinicopathological features of NPC was analyzed. The correlation among those four expression was analyzed by Spearman. The survival of NPC and independent factors of prognosis were tested by Kaplan-Meier and COX proportional hazards model respectively. The NPC group had higher positive expression rates of IGF1R, RACK1, STAT3 and Bcl-xl, and elevated expression of IGF1R, RACK1, STAT3, Bcl-xl, Bcl-2 and Bax. The lymph node metastasis (LNM) group had higher positive expression rates of IGF1R and RACK1 when compared with the non-LNM group. Patients with stage III and IV had higher positive expression rates of IGF1R, RACK1, STAT3 and Bcl-xl. There was positive correlation between expression of IGF1R and RACK1, STAT3. Such correlation was found between RACK1 and STAT3. Patients with negative expression of IGF1R, RACK1, STAT3 and Bcl-xl had higher survival rates. The risky factors of poor prognosis of NPC were positive expression of IGF1R, RACK1, STAT3 and Bcl-xl and LNM. IGF1R-RACK1-STAT3 and Bcl-xl expression correlated with the clinicopathological features and poor prognosis of NPC. This article is protected by copyright. All rights reserved

Description: Wnt signaling is hyper-activated in most of human cancers including colorectal carcinoma (CRC). Therefore, the introduction of new regulators for Wnt pathway possesses promising diagnostic and therapeutic applications in cancer medicine. Bioinformatics analysis introduced hsa-miR-103a, hsa-miR-1827 , and hsa-miR-137 as potential regulators of Wnt signaling pathway. Here, we intended to examine the effect of these human miRNAs on Wnt signaling pathway components, on the cell cycle progression in CRC originated cell lines and their expression in CRC tissues. RT-qPCR results indicated upregulation of hsa-miR-103a , hsa-miR-1827 and downregulation of hsa-miR-137 in CRC tissues. Overexpression of hsa-miR-103a and hsa-miR-1827 in SW480 cells resulted in elevated Wnt activity, detected by both Top/Flash assay and RT-qPCR analysis. Inhibition of Wnt signaling by using PNU-74654 or IWP-2 small molecules suggested that these miRNAs exerts their effect at the β-catenin degradation complex level. Then, RT-qPCR, dual luciferase assay, and western blotting analysis indicated that APC and APC2 transcripts were targeted by hsa-miR-103a , hsa-miR-1827 while, Wnt3a and β.catenin genes were upregulated. However, hsa-miR-137 downregulated Wnt3a and β.catenin genes. Further, hsa-miR-103a and hsa-miR-1827 overexpression resulted in cell cycle progression and reduced apoptotic rate in SW480 cells, unlike hsa-miR-137 overexpression which resulted in cell cycle suppression, detected by flowcytometry and Anexin analysis. Overall, our data introduced hsa-miR-103a , hsa-miR-1827 as onco-miRNAs and hsa-miR-137 as tumor suppressor which exert their effect through regulation of Wnt signaling pathway in CRC and introduced them as potential target for therapy. This article is protected by copyright. All rights reserved

Description: Cezanne, a deubiquitinating cysteine protease (DUB) belonging to A20 subgroup of ovarian tumor (OTU) protein superfamily, functions as a negative regulator of NF-κB to attenuate NF-κB activation and to restrain pro-inflammatory transcription in response to TNF receptor (TNFR) signaling. It is the first documented OTU DUB that preferably disassembles Lys11-linked polyubiquitin chains and has been shown to regulate multiple cellular events including immune signaling, cell survival and tumor progression. Previous studies showed that in response to TNF stimulation, Cezanne is recruited to the activated TNFR complex to suppress the build-up of polyubiquitinated RIP1 signal by removing Lys63 polyubiquitin from RIP1. However, how is Cezanne recognized and recruited to TNFR complex is not clear yet. In this study, we characterized a ubiquitin-associated (UBA) domain in the N-terminal region of Cezanne and proved its activity to bind Lys63 polyubiquitin chain. By constructing a series of truncated and site-specific point mutants, we further localized the crucial binding sites for Lys63 polyubiquitin chains at Leu9 and Ser10 sites of Cezanne UBA domain. Mutation at these sites disrupted the recruitment of Cezanne to activated TNFR complex and dramatically reduced the inhibition of NF-κB activation by Cezanne. Our study demonstrated that the N-terminal UBA domain is crucial for the function of Cezanne during NF-κB activation. Cezanne is recognized and recruited into activated TNFR complex by specifically binding to polyubiquitinated signaling proteins after TNF stimulation through its N-terminal polyubiquitin binding site. This study sheds light on the molecular mechanism of negative regulation of NF-κB activation by Cezanne. This article is protected by copyright. All rights reserved

Description: The objective of this study was to explore the role of miRNAs in the control of HPH as well as molecular mechanism underlying. Computational analysis and luciferase assay were carried out to search the target gene of miR-26b. Luciferase assay, RT-PCR and western-blot analysis was performed to test interaction among hypoxia, miR-26b, SRF and CTGF. MiR-26b was significantly downregulated; meanwhile, CTGF and SRF were significantly upregulated in HPH rat model. Using computational analysis, CTGF was found to be a virtual target gene of miR-26b, and only cell transfected with vectors containing wild-type CTGF 3'UTR and miR-26b showed a lower luciferase activity than scramble control. Hypoxia significantly inhibited miR-26b promoter, and promoted SRF promoter. Meanwhile, hypoxia had no effect on CTGF promoter. In addition, SRF promoted the promoter of CTGF. MiR-26b was significantly downregulated; meanwhile, CTGF and SRF were upregulated in PASMCs exposed to hypoxia. In addition, miR-26b and SRF siRNA, but not CTGF siRNA, significantly inhibited SRF expression. Meanwhile, miR-26b, SRF siRNA and CTGF siRNA significantly inhibited CTGF expression in hypoxia-treated cell. PASMCs treated with hypoxia showed higher cell viability and higher percentage cells in S phase than the control, which could be reversed by miR-26b, SRF siRNA and CTGF siRNA transfection. These findings suggested that hypoxia induced miR-26b inhibition and SRF and CTGF upregulation in HPH rat model. CTGF mediated hypoxia-induced regulation of miR-26b and SRF in proliferation of PASMCs, which indicated that hypoxia-induced miR-26b inhibition contributed to the pathogenesis of HPH via CTGF. This article is protected by copyright. All rights reserved

Description: Naringenin (NAR) is a natural predominant flavanone and has a wide range of pharmacological activities. The aim of this study was to investigate the protective mechanisms of NAR on RANKL-induced osteoclastogenesis and osteoclast bone resorption. T cells were divided into 4 groups under different concentrations of NAR (0, 25, 50, 100 µM). CD4 + T cell subsets in different groups were evaluated by flow cytometry. TRAP staining, pit formation assays and F-actin ring immunofluorescent staining were performed. In addition, gene expression of osteoclast-specific markers was analyzed by qPCR and western blot. Our results showed that compared with the control group, there were relatively fewer Th1 and Th17 cells and more Th2 cells and Treg cells in the NAR groups. Besides, the number of TRAP-positive multinucleated osteoclasts, the areas of bone resorption pits and the size and number of F-actin rings were notably decreased in the bone marrow macrophages (BMMs) treated with T-cell supernatant containing NAR. Moreover, NAR treatment dramatically reduced the expressions of cathepsin K, c-Fos, DC-STAMP, NFATc1, TRAP and V-ATPase d2 at mRNA and protein levels. However, these effects were abolished by adding a neutralizing antibody against IL-4. In conclusion, NAR suppressed RANKL-induced osteoclastogenesis and osteoclast bone resorption by promoting the release of IL-4 from T cells. This article is protected by copyright. All rights reserved

Description: Anoikis is known as a special type of programmed cell death which occurs in response to loss of correct cell- extracellular matrix (ECM) connections. This is process could be as pivotal event in normal development and tissue homeostasis and found as important mechanism in cancer invasiveness and metastasis. The persistent infection with oncoviruses including EBV (Epstein Bar virus), HPV (Human Papillomaviruses), HBV (Hepatitis B virus), KSHV (Human herpesvirus 8), HTLV-1 (Human T-lymphotropic virus-1) and HCV (Hepatitis C virus) accounted as one of main risk factor for cancer progression. Some of them play critical roles in metastasis, especially in anoikis resistance which could contribute to metastasis of tumor cells. The better understanding of effects of oncoviruses on anoikis could contribute to finding of effective therapeutic platforms for treatment of virus-associated cancers. This paper highlighted effects of these oncoviruses on anoikis protection in cancer. This article is protected by copyright. All rights reserved

Description: Methylation of arginine residues is an important modulator of protein function that is involved in epigenetic gene regulation, DNA damage response and RNA maturation, as well as in cellular signaling. The enzymes that catalyze this post-translational modification are called protein arginine methyltransferases (PRMTs), of which PRMT1 is the predominant enzyme. Human PRMT1 has previously been shown to occur in seven splicing isoforms, which are differentially abundant in different tissues, and have distinct substrate specificity and intracellular localization. Here we characterize a novel splicing isoform which does not affect the amino-terminus of the protein like the seven known isoforms, but rather lacks exons 8 and 9 which encode the dimerization arm of the enzyme that is essential for enzymatic activity. Consequently, the isoform does not form catalytically active oligomers with the other endogenous PRMT1 isoforms. Photobleaching experiments reveal an immobile fraction of the enzyme in the nucleus, in accordance with earlier results from our laboratory that had shown a tight association of inhibited or inactivated PRMT1 with chromatin and the nuclear scaffold. Thus, it apparently is able to bind to the same substrates as catalytically active PRMT1. This isoform is found in a variety of cell lines, but is increased in those of cancer origin or after expression of the EMT-inducing transcriptional repressor Snail1. We discuss that the novel isoform could act as a modulator of PRMT1 activity in cancer cells by acting as a competitive inhibitor that shields substrates from access to active PRMT1 oligomers. This article is protected by copyright. All rights reserved

Description: Thymosin beta-4 (Tβ4) is a ubiquitous protein, which has been suggested to regulate multiple cell signal pathways and a variety of cellular functions. However, the role Tβ4 plays in the cardiac microvascular endothelial cells (CMECs) under myocardial ischemia/reperfusion injury (MIRI) is currently unknown. Here we investigated the effects of Tβ4 on hypoxia/reoxygenation (H/R) induced CMECs injury and its potential molecular mechanism. Cultured CMECs were positively identified by flow cytometry using antibody against CD31 and VWF/Factor VIII, which are constitutively expressed on the surface of CMECs. Then the reduced level of Tβ4 was detected in H/R-CMECs by RT-qPCR. In order to determine the effects of Tβ4 on H/R-CMECs, we transfected the overexpression or silence vector of Tβ4 into CMECs under H/R condition. Our results indicated that H/R treatment could reduce proliferation, increased apoptosis, adhesion and ROS production in CMECs, which were attenuated by Tβ4 overexpression or aggravated by Tβ4 silence, implying Tβ4 is able to promote CMECs against H/R-induced cell injury. Furthermore, the microRNA 200a (miR-200a) level was also increased by Tβ4 in H/R-CMECs or reduced by Tβ4 siRNA. To investigated the mechanism of protective effects of Tβ4 on CMECs injury, the miR-200a inhibitor was transfected into H/R-CMECs. The results indicated that inhibition of miR-200a inversed the protection of Tβ4 on H/R-CMECs, specifically including cell proliferation, cell adhesion, cell apoptosis and ROS production, as well as nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation. In conclusion, our results determined that Tβ4 attenuated H/R induced CMECs injury by miR-200a-Nrf2 signaling. This article is protected by copyright. All rights reserved

Description: Cutaneous squamous cell carcinoma (cSCC) is a malignancy of keratinocyte-derived skin tumor, which is regarded as the second most common skin cancer worldwide. Accumulating evidence has established that microRNAs (miRNAs) can play a critical role in tumor initiation, progression and metastasis including cSCC. Abnormal expression of hsa-miR-142-5p has been elaborated in various tumors. Nevertheless, its expression and function in the development of cSCC remain unclear. In our study, we found that the expression of hsa-miR-142-5p in cSCC cells were greatly overexpressed compared to human benign epidermal keratinocyte cells. Moreover, inhibited hsa-miR-142-5p can repress cSCC cell growth and induce apoptosis while upregulated hsa-miR-142-5p exhibited a reverse phenomenon. Recently, cancer stem cells (CSCs) which possess the ability of self-renewal and proliferation and are able to produce cancer cells have been widely reported. However, the correlation between hsa-miR-142-5p and CSCs in cSCC is still unknown. Interestingly, we observed that overexpressing hsa-miR-142-5p can induce CSC-like properties in cSCC via activating Wnt signaling. In addition, the luciferase reporter assay data and bioinformatics analysis demonstrated that hsa-miR-142-5p can target the 3'UTR of PTEN mRNA. Taken these together, we draw a conclusion that hsa-miR-142-5p can trigger cancer stem cell-like properties of cSCC through inhibition of PTEN. Our findings may provide hsa-miR-142-5p as a new therapeutic target for cSCC. This article is protected by copyright. All rights reserved

Description: Breast cancer (BC) is one of the leading causes of cancer deaths worldwide and the most common cancer among women. In our previous study, we revealed that lncRNA TP73-AS1 promotes breast cancer cell proliferation through directly binding to miR-200a. Herein, we evaluated the effect of TP73-AS1 in breast cancer cell invasion and migration, and further demonstrated the direct binding between TP73-AS1 and miR-200a, between miR-200a and 3'UTR of ZEB1, an essential metastasis-related transcription factor. TP73-AS1 promoted ZEB1 expression via competing with ZEB1 3'UTR for miR-200a binding. Moreover, ZEB1 could bind to the promoter region of TP73-AS1 to activate its expression. TP73-AS1 and ZEB1 expression was up-regulated, whereas miR-200a expression was down-regulated in breast cancer tissues. Taken together, we demonstrated a TP73-AS1/miR-200a/ZEB1 regulating loop in breast cancer cells, which promote cancer cell invasion and migration through regulating E-cadherin and Twist expression. Suppressing TP73-AS1 expression to rescue miR-200a expression, thus to inhibit ZEB1 and Twist expression and up-regulate E-cadherin might improve breast cancer cell invasion and migration. This article is protected by copyright. All rights reserved

Description: Angiogenesis of liver sinusoidal endothelial cells (LSECs) accompanies with hypoxia in liver fibrosis and they are of mutual promotion, which has raised wide concern. Here we established murine model of liver fibrosis and found that oroxylin A (40 mg/kg) could ameliorate angiogenesis in liver fibrosis may related to hypoxia inducible factor 1α (HIF-1α). The underlying mechanism was futher investigated by isolating and culturing murine primary LSECs. Hypoxia induced vascular endothelial growth factor A (VEGF-A), angiopoietin 2 (Ang-2) and platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) elevated in LSECs were reduced by oroxylin A or acriflavine (ACF, an HIF-1α inhibitor), indicating HIF-1α involved the angiogenesis of LSECs. Interference with YAP significant downregulated the protein expression of HIF-1α and VEGF-A, while YAP plasmid exhibited an opposite effect. We next found that oroxylin A inhibited hypoxia-induced nuclear translocation of YAP, which may influence the accumulation of HIF-1α and subsequently decrease transcription of downstream target gene including VEGF-A and Ang-2, thereby exerting an anti-angiogenic activity. This article is protected by copyright. All rights reserved

Description: Osteosarcoma is a malignant tumor of the skeletal system. The zinc finger transcription factor ZIC2 has been reported to be highly expressed in human cancers. The present study evaluated the effects of ZIC2 and the possible underlying mechanisms in the human osteosarcoma cells. The expression levels of ZIC2 in human fetal osteoblastic cell line (hFOB1.19), osteosarcoma cell lines (U-2OS, SaoS2 and MG63), normal bone tissue, and osteosarcoma tumor were analyzed by western blot, and real-time quantitative RT-PCR (qRT-PCR). Osteosarcoma cells with either overexpressed ZIC2 or suppressed ZIC2 were analyzed to determine cell viability, colony formation, cell invasion. The expressions of SHIP2 and PI3K/AKT signal pathway-related proteins were analyzed by western blot and qRT-PCR. We first showed that ZIC2 is highly expressed in osteosarcoma cells and tissues. Then we demonstrated that overexpression of ZIC2 promoted viability,migration and invasion of osteosarcoma cells, whereas suppression of ZIC2 showed opposite effects. Furthermore, SHIP2 expression was negatively regulated by ZIC2. Importantly, ZIC2 overexpression activated the PI3K/AKT signal pathway; however, overexpressed SHIP2 inhibited these effects. Lastly, we showed that activation of the PI3K/AKT signal pathway is essential for the effects of ZIC2 on osteosarcoma cells, as the effects of ZIC2 on the osteosarcoma cells were reversed by a PI3K/AKT inhibitor. Overall, ZIC2 is highly expressed in osteosarcoma cells and tissues, and its overexpression promotes viability, invasion of osteosarcoma cells via SHIP2 suppression and PI3K/AKT activation. Thus ZIC2 can be considered as a novel drug target for osteosarcoma management. This article is protected by copyright. All rights reserved

Description: ABSTRACT Biliary epithelial cells (BEC) are closely related to some immune regulatory bile duct diseases. However, the complexity and polymorphism of the morphology and function of bile duct cells have hindered further investigation. Therefore, the aim of this study is to investigate how interleukin-6 (IL-6) affects the migration, cellular senescence and apoptosis of human intrahepatic biliary epithelial cells (HIBECs). The HIBECs were stimulated by different concentrations of IL-6 (0, 5, 10, 15 and 20 ng/ml, respectively). Transwell assay was performed in order to measure the migration abilities, positive β-Galactosidase staining for the cellular senescence of HIBECs, MTT assay for changes of proliferation after IL-6 treatment and flow cytometry for cell cycle and apoptosis. The reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting were conducted in order to detect the mRNA and protein expressions of epithelial-mesenchymal transition (EMT) markers in HIBECs. In comparison to the 0 ng/ml group, in the 5, 10, 15 and 20 ng/ml groups, a significant increase in the number of migratory HIBECs, proliferation, along with mRNA and protein expressions of EMT markers was observed. While the mRNA and protein expressions of epithelial markers, the number of β-Galactosidase positive staining cells, as well as apoptosis rate of HIBECs dramatic decreased. Further, the aforementioned changes were significantly more evident in the 15 and 20 ng/ml groups in comparison to the 5 and 10 ng/ml groups. IL-6 may stimulate EMT, enhance the migration and proliferation, and inhibit apoptosis of HIBECs, thus delaying cellular senescence. This article is protected by copyright. All rights reserved

Description: Melanoma represents an ever-increasing problem in the western world as incidence rates continue to climb. Though manageable during early stages, late stage metastatic disease is highly resistant to current intervention. We have previously shown that gamma-interferon-inducible lysosomal thiol-reductase (GILT) enhances HLA class II antigen processing and immune detection of human melanoma cells. Here we report that GILT expression inhibits a potential target, paired box-3 (PAX-3) protein, in late stage human metastatic melanoma. We also show that GILT transfection or induction by IFN-?, decreases PAX-3 protein expression while upregulating the expression of Daxx, which is also a repressor of PAX-3. Confocal microscopic analysis demonstrated that GILT co-localizes with PAX-3 protein, but not with Daxx within melanoma cells. Immunoprecipitation and immunoblotting studies suggest that GILT expression negatively regulates PAX-3 through the autophagy pathway, potentially resulting in increased susceptibility to conventional treatment in the form of chemotherapy or radiotherapy. While high-dose radiation is a common treatment for melanoma patients, our data suggest that GILT expression significantly increased the susceptibility of melanoma cells to low-dose radiation therapy via upregulation of tumor suppressor protein p53. Overall, these data suggest that GILT has multiple roles in inducing human melanoma cells as better targets for radiation and immunotherapy. This article is protected by copyright. All rights reserved

Description: Our study was performed to elucidate how SOCS-1/3 silencing suppresses renal interstitial fibrosis (RIF) by alleviating renal tubular damage in rat models affected by hydronephrosis. Male Wistar rats were randomly selected to establish hydronephrosis rat model, after which all rats were classified into normal, model, negative control (NC), siRNA-SOCS-1, siRNA-SOCS-3, and siRNA-SOCS-1 + siRNA-SOCS-3 groups. The levels of urine protein, serum creatinine (Scr), and blood urea nitrogen (BUN) were detected. ELISA was performed to determine levels of cystatin (CysC), β2-microglobulin (β2-MG), interleukin (IL)-6, and tumor necrosis factor (TNF)-α. RT-qPCR and western blotting were used for mRNA and protein expressions of SOCS-1, SOCS-3, α-smooth muscle actin (α-SMA), and transforming Growth Factor (TGF)-β1. Compared with the normal group, the levels of Scr, BUN, urine protein, NAG, CysC, β2-MG, IL-6, and TNF-α were increased in other groups, as well as elevated mRNA and protein expressions of SOCS-1, SOCS-3, α-SMA, and TGF-β1. The siRNA-SOCS-1, siRNA-SOCS-3, and siRNA-SOCS-1 + siRNA-SOCS-3 groups were found with decreased levels of Scr, BUN, urine protein, NAG, CysC, β2-MG, IL-6, and TNF-α, as well as mRNA and protein expressions of SOCS-1, SOCS-3, α-SMA and TGF-β1, including positive rates of SOCS-1 and SOCS-3 proteins in comparison with the model and NC groups. In comparison with the siRNA-SOCS-1 and siRNA-SOCS-3 groups, the siRNA-SOCS-1 + siRNA-SOCS-3 group exhibited decreased levels of Scr, BUN, urine protein, NAG, CysC, β2-MG, IL-6, and TNF-α. Our study demonstrated that silencing of SOCS-1/3 may suppress RIF by alleviating the renal tubular damage in rat models affected by hydronephrosis. This article is protected by copyright. All rights reserved

Description: ABSTRACT The cell-based therapies could be potential methods to treat damaged cartilage tissues. Instead of native hyaline cartilage, the current therapies generate mainly weaker fibrocartilage-type of repair tissue. A correct microenvironment influences the cellular phenotype, and together with external factors it can be used, e.g., to aid the differentiation of mesenchymal stem cells to defined types of differentiated adult cells. In this study, we investigated the effect of long-term exposure to 5% low oxygen atmosphere on human chondrosarcoma HCS-2/8 cells. This atmosphere is close to normal oxygen tension of cartilage tissue. The proteome was analyzed with label-free mass spectrometric method and further bioinformatic analysis. The qRT-PCR method was used to gene expression analysis, and ELISA and dimethylmethylene blue assays for type II collagen and sulfated glycosaminoglycan measurements. The hypoxic atmosphere did not influence cell proliferation, but enhanced slightly ACAN and COL2A1 gene expression. Proteomic screening revealed a number of hypoxia-induced protein level responses. Increased ones included NDUFA4L2, P4HA1, NDRG1, MIF, LDHA, PYGL, while TXNRD1, BAG2, TXN2, AQSTM1, TNFRSF1B and EPHX1 decreased during the long-term hypoxia. Also a number of proteins previously not related to hypoxia changed during the treatment. Of those S100P, RPSS26, NDUFB11, CDV3 and TUBB8 had elevated levels, while ALCAM, HLA-B, EIF1, and ACOT9 were lower in the hypoxia samples. In conclusion, low oxygen condition causes changes in the cellular amounts of several proteins. This article is protected by copyright. All rights reserved

Description: Stathmin/oncoprotein18 regulates microtubule dynamics and participates in mitotic entry and exit. We isolated stathmin as a physically interacting partner of KIFC1, a minus-end-directed kinesin functioning in bipolar spindle formation and maintenance. We found that stathmin depletion leads to multipolar spindle formation in IMR-90 normal human fibroblasts. Stathmin-depleted IMR-90 cells showed early mitotic delay but managed to undergo chromosome segregation by forming multiple poles or pseudo-bipoles. Consistent with these observations, lagging chromosomes and micronuclei were elevated in stathmin-depleted IMR-90 cells, demonstrating that stathmin is essential for maintaining genomic stability during mitosis in human cells. Genomic instability induced by stathmin depletion led to premature senescence without any indication of cell death in normal IMR-90 cells. Double knock-down of both stathmin and p53 also did not induce cell death in IMR-90 cells, while the stathmin knock-down triggered apoptosis in p53-proficient human lung adenocarcinoma cells. Our results suggest that stathmin is essential in bipolar spindle formation to maintain genomic stability during mitosis, and the depletion of stathmin prevents the initiation of chromosome instability by inducing senescence in human normal fibroblasts. This article is protected by copyright. All rights reserved

Description: Leptin, an adipokine secreted by adipose tissue, induces cell invasion and metastasis. MMP7 is a member of the matrix metalloproteinase family that plays an important role in cell invasion. Here we evaluate the possible role and underlying mechanism of MMP7 in the leptin- mediated cell invasion in ovarian cancer cell lines. All experiments were carried out in cultured SKOV3, OVCAR3 and CaoV-3 ovarian cell lines. MMP7 expression was determined using the western blot following treatment to various concentrations of leptin for defined time intervals. The activation of ERK, JNK and P38 MAP kinases were determined using western blotting. Wound healing and BD matrigel invasion assays were used to measure cell migration and invasion. The siRNA approach and pharmacological inhibitors of ERK and JNK pathway were used to confirm the receptor-dependent effect of leptin and a role for ERK and JNK pathway. Zymography assay was employed to determine MMP2 and MMP9 activation. Results show that leptin induces ERK1/2 and JNK1/2 activation and subsequently promotes MMP7 expression in SKOV3 (4.8±0.14 fold of control, p 〈 0.01) and OVCAR3 (3.1±0.19 fold of control, p 〈 0.01) ovarian cancer cell lines. These effects was reversed by knockdown of OB-Rb and/or pre-incubation with PD98059 (ERK1/2 inhibitor), SP600125 (JNK1/2 inhibitor). Gelatin zymography showed that MMP7 gene silencing attenuated leptin-induced MMP9 activation in SKOV3 cell line. Taken together, our results suggest new evidences for a modulatory effect of leptin in regulation of ovarian cancer cell invasion by stimulating MMP7 expression via ERK and JNK pathways. This article is protected by copyright. All rights reserved

Description: Malignant cancers are distinguished from more benign forms of cancers by enhanced ability to disseminate. A number of factors aid the migration and invasion of malignant cancer cells. Epithelial-to-mesenchymal transition (EMT), which greatly facilitates the dissemination of cancer cells, is characterized by the loss of epithelial markers and the acquisition of mesenchymal markers thereby rendering the cells more migratory and invasive. We have previously shown that the class III lysine deacetylase SIRT1 plays a critical role curbing the metastasis of ovarian cancer cells partly by blocking EMT. Here we investigated the mechanism by which SIRT1 regulates the transcription of Claudin 5 (CLDN5), an epithelial marker gene, in ovarian cancer cells. SIRT1 activation or over-expression up-regulated CLDN5 expression while SIRT1 inhibition or depletion down-regulated CLDN5 expression. SIRT1 interacted with and deacetylated Kruppel-like factor 4 (KLF4), a known transcriptional activator for CLDN5. Deacetylation by SIRT1 promoted nuclear accumulation of KLF4 and enhanced the binding of KLF4 on the CLDN5 promoter in the nucleus. SIRT1-mediated up-regulation of CLDN5 was abrogated in the absence of KLF4. In accordance, KLF4 depletion by siRNA rendered ovarian cancer cells more migratory and invasive despite of SIRT1 activation or over-expression. In conclusion, our data suggest that SIRT1 activates CLDN5 transcription by deacetylating and potentiating KLF4. This article is protected by copyright. All rights reserved

Description: Aminoflavone (AFP 464, NSC 710464), an antitumor agent which recently entered phase II clinical trials, acts against estrogen-positive breast cancer (ER + ) . AFP 464, which has a unique mechanism of action by activating aryl hydrocarbon receptor (AhR) signaling pathway, decreased tumor size and growth rate in the estrogen dependent, Tamoxifen-sensitive spontaneous M05 mouse model. Considering that AhR has recently emerged as a physiological regulator of the innate and adaptive immune responses, we investigated whether AFP 464 modulates the immune response in our mouse model. Studies on the effect of AFP 464 on the immune system were carried in BALB/c mice bearing M05 semi-differentiated mammary adenocarcinomas that express estrogen and progesterone receptors. Splenic cells and tumor inflammatory infiltrates were studied by cytometric analyses. The modulation of splenocytes cytotoxic activity by AFP 464 was also evaluated. We further investigated the effects of AFP 464 on peritoneal macrophages by evaluating metalloproteinase, arginase and iNOS activities. We found that AFP 464 increased splenic cytotoxic activity, diminished the number of systemic and local Treg lymphocytes and MDSCs, and induced a M1 phenotype in peritoneal macrophages of M05 tumor bearing mice. Therefore, we conclude that AFP 464 modulates immune response which collaborates with its anti-tumor activity. Our results place the immune system as a novel target for this anti-cancer agent to strengthen the rationale for its inclusion in breast cancer treatment regimens. This article is protected by copyright. All rights reserved

Description: We have previously shown that the DNA replication licensing factor ORC4 forms a cage around the chromosomes that are extruded in both polar bodies during murine oogenesis, but not around the chromosomes that are retained in the oocyte or around the sperm chromatin. We termed this structure the ORC4 cage. Here, we tested whether the formation of the ORC4 cage is necessary for polar body extrusion (PBE). We first experimentally forced oocytes to extrude sperm chromatin as a pseudo-polar body and found that under these conditions the sperm chromatin did become enclosed in an ORC4 cage. Next, we attempted to prevent the formation of the ORC4 cage by injecting peptides that contained sequences of different domains of the ORC4 protein into metaphase II oocytes just before the cage normally forms. Our rationale was that the ORC4 peptides would block protein-protein interactions required for cage formation. Two out of six tested peptides prevented the ORC4 cage formation and simultaneously inhibited polar body extrusion (PBE), resulting in the formation of two pronuclei that were retained in the oocyte. Together, these data demonstrate that ORC4 oligomerization is required to form the ORC4 cage and that it is required for PBE. This article is protected by copyright. All rights reserved

Description: Glioblastoma Multiforme (GBM) is a highly malignant primary brain cancer that is associated with abysmal prognosis. The median survival of GBM patients is ∼15 months and there have not been any significant advance in therapies in over a decade, leaving treatment options limited. There is clearly an unmet need for GBM treatment. Immunotherapies are treatments based on usurping the power of the host's immune system to recognize and eliminate cancer cells. They have recently proven to be a successful strategy for combating a variety of cancers. Of the various types of immunotherapies, checkpoint blockade approaches have thus far produced significant clinical responses in several cancers including melanoma, non small-cell lung cancer, renal cancer and prostate cancer. This review focuses on the biological rationale for using checkpoint blockade immunotherapeutic approaches in primary brain cancer and an up-to-date summary of current and ongoing checkpoint inhibitors-based clinical trials for malignant glioma. In addition, we expand on new concepts for further improving checkpoint blockade treatments, with a particular focus on the advantages of using genetically engineered mouse models for studies of immunotherapies in GBM. This article is protected by copyright. All rights reserved

Description: A revolutionary approach that involves direct conversion of somatic cells into almost any other types of cells showed promising results for regenerative medicine. Currently, producing valuable cell types including neurons, cardiomyocytes and hepatocytes through direct conversion of somatic cells appear to be a feasible option for regenerative medicine. The process involves inducing the cells by chemical cocktails or by expression of different types of transcription factors. In this concept, in vitro neurogenesis considered to be able to produce neuron cells to replace damaged neurons especially in Alzheimer and Parkinson disease. However, early successful experiments followed by major drawbacks such as low differentiation efficiency in producing neurons and detection of various undesirable types of cells in the culture. Therefore, there is not a single optimized common protocol for producing high quality neurons in vitro so far. This is partly due to the lack of our understanding about the precise cellular, genetic, and molecular mechanisms underlying neurogenesis via direct conversion. In the current work, we have employed meta-analysis tools and extensive gene regulatory network analysis on the high through put gene expression data obtained from previous reprogramming protocols to identify central gene regulatory components involved in direct conversion of fibroblasts into neurons. Our results identified miR-9, miR-30 as the most important miRNA and TP53, MYC, JUN, SP1 and SMAD2 considered to be the most important transcription factors. These findings would be useful for direct targeting these hub regulatory elements in order to increase the efficacy and specificity of the conversion protocols. This article is protected by copyright. All rights reserved

Description: Numerous studies have demonstrated the role of A3 adenosine receptor (A3AR) and signaling pathways in the multiple aspects of the tumor. However, there is a little study about the function of A3AR in the biological processes of cancer stem cells (CSCs). CSCs have a critical role in the maintenance and survival of breast cancer. The aim of current study was to investigate the effect of A3AR agonist on breast cancer stem cells (BCSCs). XTT assay showed antiprolierative effect of A3AR agonist (Cl-IB-MECA) on BCSCs. Our results also demonstrated that A3AR agonist reduces mammosphere formation in a dose-dependent manner. Flow cytometry analysis showed that A3AR agonist induces G1 cell cycle arrest and apoptosis in BCSCs. Western blot assay showed that A3AR agonist inhibits the expression of cell cycle and apoptotic regulatory proteins as well as the expression of ERK1/2 and GLI-1 proteins. Finally, these findings propose that A3AR agonist induces cell cycle arrest and apoptosis in BCSCs by inhibition of ERK1/2 and GLI-1cascade. This article is protected by copyright. All rights reserved

Description: ABSTRACT Folate deficiency is a known risk factor for liver injury; however, the underlying mechanism remains unclear. In this study, we employed a high homocysteine-induced liver injury model of Apolipoprotein E-deficient ( ApoE −/− ) mice fed high-methionine diet and found that high homocysteine induced endoplasmic reticulum (ER) stress and liver cell apoptosis by downregulation of cystic fibrosis transmembrane conductance regulator (CFTR) expression; observations that were attenuated with supplementation of dietary folate. The regulation on CFTR expression was mediated by CFTR promoter methylation and trimethylation of lysine 27 on histone H3 (H3K27me3). Mechanistically, folate inhibited homocysteine-induced CFTR promoter methylation and H3K27me3, which resulted in upregulation of CFTR expression, and reduced ER stress and liver cell apoptosis. Further study showed that folate inhibited the expression of DNA methyltransferase 1 and enhancer of zeste homolog 2, downregulated the cellular concentrations of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) and upregulated the SAM/SAH ratio, leading to the inhibition of Hcy-induced DNA hypermethylation and H3K27me3 in CFTR promoter. In conclusion, our results provide insight into the protective role of folate in homocysteine-induced ER stress and liver cell apoptosis through the regulation of CFTR expression. This article is protected by copyright. All rights reserved

Description: ABSTRACT N 6 -methyladenosine (m 6 A) modification is an abundant and conservative RNA modification in bacterial and eukaryotic cells. m 6 A modification mainly occurs in the 3′ untranslated regions (UTRs) and near the stop codons of mRNA. Diverse strategies have been developed for identifying m 6 A sites in single nucleotide resolution. Dynamic regulation of m 6 A is found in metabolism, embryogenesis and developmental processes, indicating a possible epigenetic regulation role along RNA processing and exerting biological functions. It has been known that m 6 A editing involves in nuclear RNA export, mRNA degradation, protein translation and RNA splicing. Deficiency of m 6 A modification will lead to kinds of diseases, such as obesity, cancer, type 2 diabetes mellitus, infertility, developmental arrest. Some specific inhibitors against methyltransferase and demethylase have been developed to selectively regulate m 6 A modification, which may be advantageous for treatment of m 6 A related diseases. This article is protected by copyright. All rights reserved

Description: The aim of our study was to investigate the effects of miR-133a-3p on human oral squamous cell carcinoma (OSCC) cells by regulating gene COL1A1 . OSCC tissues, adjacent tongue epithelial tissues, the immortalized oral epithelial cell line HIOEC and OSCC cell lines (CAL-27, TCA-8113, SCC-4, SCC-9 and SCC-15) were used in this research. Quantitative real-time PCR (RT-qPCR) was employed to determine the expression of miR-133a-3p and COL1A1 . Dual luciferase reporter gene assay and Western blot were applied to verify the binding relationship between miR-133a-3p and COL1A1 . Functional assays were also conducted in this study, including CCK-8 assay, colony formation assay, flow cytometry analysis as well as Transwell assay. MiR-133a-3p was found low-expressed both in OSCC tissues and cells lines compared with normal tissues and cell line, respectively, whereas COL1A1 was just the opposite. The over-expression of miR-133a-3p or the down-regulation of COL1A1 suppressed the proliferation, invasion and mitosis of OSCC cells, whereas simultaneous down-regulation of miR-133a-3p and up-regulation of COL1A1 led to no significant alteration of cell activities. MiR-133a-3p could inhibit the proliferation and migration of OSCC cells through directly targeting COL1A1 and reducing its expression. This article is protected by copyright. All rights reserved

Description: Thank you for providing the authors' responses to our comments (Fallahi et al. 2017). After a careful review of their responses, we believe that the authors did not exactly answer our concerns, and instead provided blanket reasons such as “we did not understand their paper, methodology, etc.” In addition, in their response, the authors clearly tried to change their primary claims in comparison to what was written in their published paper, again without answering our questions. Therefore, we decided to write another letter, to markedly show that their conclusions are unreliable and incorrect in both their strategies to analyze data sets independently (which they are now claiming to have done) or in combination (which was their claim in the previous paper and for which we addressed as part of a number of problems in our previous letter). This article is protected by copyright. All rights reserved

Description: Although, the precise host defence mechanism(s) is not completely understood, T cell-mediated immune responses is believed to play a pivotal role in controlling parasite infection. Here we target the stage dependent over expressed gene. Here, the consensus based computational approach was adopted for the screening of potential major histocompatibility complex class I restricted epitopes. Based on the computational analysis and previously published report, a set 19 antigenic proteins derived from Leishmania donovani were screened for further characterization as vaccine candidates. A total of 49 epitopes were predicted, which revealed a comprehensive binding affinity to the 40 different MHC class I supertypes. Based on the population coverage and HLA cross presentation, nine highly promiscuous epitopes such as LTYDDVWTV (P1), FLFPQRTAL(P2), FLFSNGAVV (P3), YIYNFGIRV (P4), YMTAAFAAL (P5), KLLRPFAPL (P6), FMLGWIVTI (P7), SLFERNKRV (P8) and SVWNRIFTL (P9) which have either a high or an intermediate TAP binding affinity were selected for further analysis. Theoretical population coverage analysis of polytope vaccine (P1-P9) revealed more than 92% population. Stimulation with the cocktail of peptide revealed a proliferative CD8 + T cell response and increased IFN-γ production. An upregulated NF-κB activity is thought to be play a pivotal role in T cell proliferation against the selected peptide. The Th1-type cytokine profile (presence of IFN-γ and absence of IL-10) suggests the potentiality of the cocktail of epitope as a subunit vaccine against leishmaniasis. However, the efficiency of these epitopes to trigger other Th1 cytokines and chemokines in a humanized mice model could explore its plausibility as a vaccine candidate. This article is protected by copyright. All rights reserved