ALBERT

Description: Background Circular RNAs (circRNAs), a novel family of non-coding RNAs, have crucial roles in cancer progression. Conventional research is mainly focus on nuclear genome derived circRNAs (nu-circRNAs). The biological and clinical characteristics of mitochondrial genome derived-circRNAs (mt-circRNA) remains largely unknown, especially in chronic lymphocytic leukemia (CLL), the most prevalent incurable B-cell neoplasm in western countries. Lack of convenient and reliable clinical biomarkers is a hindrance in monitoring the progression of CLL. It is in urgent need of screening effective new biomarkers and exploring potential therapeutic targets associated with CLL initiation and progression. Recent studies have shown that circRNAs are enriched and stable in serum exosomes. However, the biological mechanisms of exosomal circRNAs remain unclear. In this study, we attempted to identify the novel characteristics of a mt-circRNA mc-COX2, which was abundant in plasma exosomes and could be involved in the progression of CLL. Since CLL patients have specific expression features of exosomal circRNAs in plasma, the function of the circRNAs and their clinical significance is urged to be explored. Methods Firstly, to unveil circRNAs expression profiles in CLL, plasma samples from five treatment-naive CLL patients and five age-/sex-matched healthy donors (HDs) were collected for circRNA microarray analyses. Northern blot and RNA- FISH were conducted to verify the existence of circRNAs in mitochondria. qPCR and other functional analysis such as RNase R, actinomycin D and RIP experiments were carried out to demonstrated the clinical and biological characteristics of mc-COX2, one of the mt-circRNAs. Cell apoptosis ability was determined by FCM. Moreover, electron microscope, partical size analysis, FCM and Western blot were used to explore the existence of exosomes and q-PCR analysis was performed to detect the expression of mc-COX2. Results Mt-circRNAs were highly expressed in CLL patients plasma (Figure A, B). Herein, we reported a novel circRNA named as mc-COX2 which was generated from the COX2 gene on mitochondrial genome by back-splicing and closely related to prognosis of CLL patients (Figure C). The enrichment of mc-COX2 in the mitochondria was further confirmed by RNA-FISH (Figure D). Northern blot was performed using head-to-tail probe of mc-COX2 and the results showed that mc-COX2 was detectable within the splice sites (Figure E). Notably, obviously different from nu-circRNAs, mc-COX2 showed lower stability with lower tolerance to RNase R and actinomycin D, but it was much more stable compared with linear RNAs (Figure G, F). And mc-COX2 cannot bind to AGO2 protein, suggesting that it probably function via other mechanisms instead of acting as ceRNA (Figure H). Furthermore, the up-regulated expression of mc-COX2 was positively associated with leukemogenesis and worse survival of CLL patients (Figure I, J). CLL patients with TP53 deletion rather than mutation displayed higher expression of mc-COX2 (Figure K). The endogenous reduction of mc-COX2 could induce cell apoptosis (Figure L). In addition, we indicated that mc-COX2 was highly enriched in exosomes, by which circRNAs could enter the circulation and be readily measured in the serum (Figure M). Moreover, the existence of mc-COX2 in plasma suggests that mc-COX2 may serve as a potentially novel prognosis biomarker for CLL patients, guiding targeted therapy in clinic. Conclusions In summary, we demonstrated the existence and clinical significance of mc-COX2, a novel class of circRNA species abundant in CLL plasma exosomes for the first time, which was distinct from nu-circRNAs. Furthermore, the specific high expression of mc-COX2 in CLL plasma which was strongly correlated with P53 deletion, can indicate worse prognosis of CLL patients. Taken together, our study not only identifies a novel circRNA which may serve as a new "liquid biopsy" biomarker for CLL prognosis but also expands the current knowledge regarding molecular mechanisms of circRNAs, providing potential diagnostic and therapeutic implications for CLL. It would be of great interest to explore the biogenesis of mt-circRNAs and their impact on mitochondrial function in future studies. Figure Disclosures No relevant conflicts of interest to declare.

Description: Objective Chronic lymphocytic leukemia (CLL) is a heterogeneous disease and characterized by the accumulation of monoclonal B lymphocytes in blood, bone marrow and lymphoid tissues. Although diagnosis and therapy have been developing, several high-risk features for CLL patients have been identified, only few of them are well-characterized and clinically actionable. IGHV mutation status has been confirmed as a valuable independent prognostic factor. However, it is of vital importance to explore alternative markers because the detection method of IGHV mutation is expensive and time consuming, which limits its application in the routine laboratory diagnosis process. Thus, further exploration of effective diagnostic biomarkers for CLL are worthy and important. Circular RNAs (circRNAs), a novel family of non-coding RNAs, have been described as critical regulators of gene expression and are stable in exosomes. However, the compelling role of circRNAs in mediating CLL progression was still poorly understood. In this study, we attempted to identify a novel circRNA which could serve as a plasma biomarker and explore its molecular mechanism and clinical significance in CLL. Methods To analyse circRNAs expression profiles in CLL, plasma samples from three untreated CLL patients and five normal donors were collected for circRNA microarray. Then, we validated and analysed the clinical characteristics of candidate circRNAs in a larger cohort of patients (137 CLL patients vs. 50 healthy donors, age- and sex-matched). The receiver-operating characteristic (ROC) curve analysis was performed to evaluate the potential diagnostic value of circ-RPL15 as a biomarker for CLL, plasma and PBMC samples from multiple lymphomas were collected for qRT-PCR detection as well. The oncogenic functions of circ-RPL15 were further measured in CLL cell lines (MEC-1 and JVM-3) by performing RNA interfering (RNAi), immunofluorescence (IF) staining, CCK8 assay and flow cytometry. Moreover, we explored the molecular mechanisms of circ-RPL15 and verified the interactions among POLR2A, circ-RPL15, miR-146b-3p and RAF1 by performing RNA-FISH, ChIP, RIP, dual-luciferase reporter assay and Western blotting. Results A total of 3,656 circRNAs were identified, among which 71 circRNAs were dysregulated in CLL patients ( p≤0.05) and circ-RPL15 showed the most significantly high expression level and was finally selected for further investigation. Circ-RPL15 was abundantly present in exosomes and was significantly higher expressed in CLL compared with normal and other lymphomas. Moreover, we found that circ-RPL15 was highly relevant to IGHV region mutation status, which has long been considered as an important prognostic indicator of CLL. Patients without IGHV mutation presented a higher circ-RPL15 levels and the ROC curve showed that circ-RPL15 could be a good alternative to IGHV status with AUC values of 0.86. Kaplan-Meier analysis showed that CLL patients with circ-RPL15 high expression had a significantly poorer OS than those with low expression of circ-RPL15 (Figure A-F). Next, we unraveled an original mechanism in CLL that up-regulated circ-RPL15 was mainly enriched in the cytoplasm, acted as a "sponge" of miR-146b-3p. CCK8 assay and flow cytometry analysis showed that the cell viability of CLL cell lines were evidently decreased after RNAi of circ-RPL15 and this result could be reversed by miR-146b-3p inhibitor transfection. Western blot suggested a critical role of circRPL15- sponged miR-146b-3p in the regulation of RAS/RAF1/MEK/ERK pathway (Figure G-L). Furthermore, we identified circ-RPL15, a circRNA derived from exon 2 and partial exon 3 of RPL15 pre-mRNA which is located on chromosome 3p24.2, was negatively regulated by transcription factor POLR2A and delivered by exosomes into CLL patient plasma (Figure M,N). Conclusion In this study, we uncovered a novel oncogenic biomarker for CLL patients, which contributes to promoting CLL progression and is associated with poor outcome. The higher expression of circ-RPL15 was positively correlated with IGHV mutation status which is crucial in the evaluation of CLL prognosis. The miR-146b-3p mediated suppression of RAS/RAF1/MEK/ERK pathway could be alleviated by circ-RPL15 up-regulation in CLL. Our results revealed that circ-RPL15 may represent a promising plasma biomarker for the diagnosis and prognosis of CLL as well as a reliable surrogate of IGHV mutation status. Figure Disclosures No relevant conflicts of interest to declare.

Description: Aims Down syndrome (DS) is the most frequent single cause of human birth defects and intellectual disability (ID). Its etiology has been known for over 50 years, DS is caused by trisomy of chromosome 21 (Ts21). In addition, there are more evidences in clinical show that patients of DS are also have high incidence of leukemia, but its underlying mechanisms have yet to be discovered. Elucidation of these mechanisms has been hindered by the difficulties in isolating and expanding enough hematopoietic stem cells (HSCs) from the patients. Circular RNAs (CircRNAs) are a novel type of endogenous noncoding RNAs that reported to play important roles in biological and pathological processes. Our present study aims to investigate the pathogenesis of Down Syndrome prone to leukemia using iPSCs from DS patients and the modulation mechansim of key target gene and circRNAs. Methods To overcome the limitation of clinical sample resources, we developed Ts21-induced pluripotent stem cells (iPSCs) from mononuclear cells using Episomal vectors which highly express Oct4,Sox2 and Klf4. Under certain conditions, these Ts-21 specific iPSCs could be further induced into any cell types, including HSCs. Next, we induced the patient-specific iPSCs differentiate into HSCs under serum free and feeder-free condition in vitro. By using flow cytometry, qRT-PCR and CFU assays, we have made well comparisons between HSCs derived from TS-21 iPSCs and control iPSCs, and expolre the pathological feature and mechanisms. By performing circRNA-seq analysis, we identified key circRNA and its host gene regulatory networks in DS with hematopoietic disorder. On the basis of that, we can further search for the potential drug targets that effective against leukemia of DS patients. Results We show here by gene sequencing, karyotyping and immunohistochemistry staining, the initial characterization of the patient-specific induced pluripotent stem are confirmed to be fully reprogrammed and they still carry the original mutation. These cells can be used for screening approaches and can be modified for loss- and gain-of-function strategies to identify pathways and test candidate mechanisms. We have observed that myeloid differentiation potential of HSCs derived from DS patients was significantly higher than normal patinets. DS-iPSCs exhibited a 2-4 fold increase in a population of CD43+/GPA+ hematopoietic cells, accompanied by increased multilineage colony-forming potential in CFU assays.During this process, has_circ_0082802 was significantly up-regulated. After knockdown of has_circ_0082802 using shRNAs, the erythroid differentiation level as well as cell proliferation were decreased. The potential modulation mechansim of them will be further explored. Conclusion We established the cellular models of DS patients and derived differentiation into different cell types in vitro, which has the similar function with patient HSCs. On the basis of that, we found has_circ_0082802 may play important role in erythroid differentiation. We can further search for the potential drug targets that effective against leukemia of DS patients. This approach would provide a powerful cell resource for clinical research and a useful model for the study of the mechanisms of DS-AML Disclosures No relevant conflicts of interest to declare.

Description: Background Acute erythroleukemia (AEL) is defined as an distinct subtype (

Description: Circular RNA (circRNA) is a novel regulatory non-coding RNA and participates in diverse physiological and pathological processes. However, the structures and molecular mechanisms of circRNAs remain unclear. In this study, taking advantage of openly databases and bioinformatics analysis, we observed lots of internal complementary base-pairing sequences (ICBPS) existed in plenty of circRNAs, especially in extremely long circRNAs (el-circRNAs, 〉 5,000 nt). The result indicated that circRNA may not be a simple circular structure. In addition, we put forward the hypothesis of “open-close effect” in the transition for specific circRNA from normal state to morbid state. Taken together, our results not only expand the knowledge of circRNAs, but also highlight the potential molecular mechanism of circRNAs.