The TNFα-induced miR-18a activates rheumatoid arthritis synovial fibroblasts through a feedback loop in NF-κB signaling

摘要

Objective: To elucidate whether the microRNA (miRNA) cluster miR-17-92 contributes to the activated phenotype of rheumatoid arthritis synovial fibroblasts (RASF). Methods: RASF were stimulated with tumor necrosis factor alpha (TNFα? and expression and regulation of the miR-17-92 cluster were studied using quantitative real-time PCR (qPCR) and promoter activity assays. RASF were transfected with single precursor molecules of miRNAs from miR-17-92 and the expression of matrix-degrading enzymes and cytokines was measured by qPCR and enzyme-linked immunosorbent assay. Potential miRNA targets were identified by computational prediction and validated using reporter gene assays and Western blot. The activity of NF-κB signaling was determined by reporter gene assay. Results: We found that TNFα induces the expression of miR-17-92 in RASF in a nuclear factor kappa B (NF-κB)-dependent manner. Transfection of RASF with precursor molecules of single members of miR-17-92 revealed significantly increased expression levels of matrix-degrading enzymes, proinflammatory cytokines and chemokines in pre-miR-18a-transfected RASF. Using reporter gene assays we identified the NF-κB pathway inhibitor TNFα-induced protein 3 (TNFAIP3) as a new target of miR-18a. In consequence, pre-miR-18a-transfected RASF showed stronger activation of NF-κB signaling, both constitutively and in response to TNFα-stimulation. Conclusion: Our data suggest that the miR-17-92-derived miR-18a contributes to cartilage destruction and chronic inflammation in the joint through a positive feedback loop in NF-κB signaling with concomitant upregulation of matrix-degrading enzymes and inflammatory mediators in RASF. © 2012 American College of Rheumatology.