Publication Date:
2011-11-18
Description:
Abstract 774 The most aggressive subtype of diffuse large B-cell lymphoma (DLBCL), ABC-DLBCL, is characterized by constitutive activation of NF-κB signals, in association with somatic mutations in regulators of this pathway. However, in a large fraction of cases the molecular basis for the elevated NF-κB activity is unclear. MicroRNAs (miRNA) play a central role in immune function, in part by modulating the NF-κB system; yet, their role in the NF-κB deregulation found in DLBCL is unknown. To address this issue, we created a copy number/expression map of miRNAs in ∼100 DLBCLs, and integrated these data with miRNA target prediction models. This supervised strategy showed that miR-125a and miR-125b, which we found to be amplified/overexpressed in ∼ 30% of DLBCLs, are predicted to target TNFAIP3 (A20) via two fully conserved binding sites. A20 is an ubiquitin editing enzyme that negatively regulates NF-κB complexes and acts as a lymphoma suppressor gene, suggesting that its abnormal downregulation by miRs-125a/b may impinge on DLBCL pathogenesis. To validate these initial findings, we stably expressed miR-125a and miR-125b in multiple DLBCL cell lines, and used western blots to show that both miRNAs downregulate A20. In parallel assays, we used anti-miR oligos and miRNA sponge constructs in cell lines expressing high levels of miRs-125a/b, and found that their inhibition upregulates A20 levels. To study the interaction between miRs-125a/b and A20, we created reporter constructs with A20 sequences wild-type (WT) or mutated for the miR-125 binding sites; luciferase activity of the WT (but not the mutant) constructs was inhibited by miRs-125a/b confirming that they directly target A20. We hypothesized that miRs-125a/b-mediated inhibition of A20 activates NF-κB in DLBCL. Indeed, phosphorylation and degradation of the NF-κB inhibitory protein IκBα was markedly enhanced in DLBCL cell lines ectopically expressing miRs-125a/b, in comparison to their isogenic counterparts lacking these miRNAs. Further, nuclear accumulation of RelA was also elevated in miRs-125a/b expressing cells. Finally, we used a NF-κB reporter construct, to show that DLBCL cells stably expressing miRs-125a/b had significantly higher NF-κB activity (p
Print ISSN:
0006-4971
Electronic ISSN:
1528-0020
Topics:
Biology
,
Medicine
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