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Abstract

Objective

To elucidate whether the microRNA (miRNA) cluster miR-17–92 contributes to the activated phenotype of rheumatoid arthritis synovial fibroblasts (RASFs).

Methods

RASFs were stimulated with tumor necrosis factor α (TNFα), and the expression and regulation of the miR-17–92 cluster were studied using real-time quantitative PCR (PCR) and promoter activity assays. RASFs were transfected with single precursor molecules of miRNAs from miR-17–92 and the expression of matrix-degrading enzymes and cytokines was measured by quantitative PCR and enzyme-linked immunosorbent assay. Potential miRNA targets were identified by computational prediction and were validated using reporter gene assays and Western blotting. The activity of NF-κB signaling was determined by reporter gene assays.

Results

We found that TNFα induces the expression of miR-17–92 in RASFs in an NF-κB–dependent manner. Transfection of RASFs with precursor molecules of single members of miR-17–92 revealed significantly increased expression levels of matrix-degrading enzymes, proinflammatory cytokines, and chemokines in precursor miR-18a (pre-miR-18a)–transfected RASFs. Using reporter gene assays, we identified the NF-κB pathway inhibitor TNFα-induced protein 3 as a new target of miR-18a. In addition, pre-miR-18a–transfected RASFs 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 up-regulation of matrix-degrading enzymes and mediators of inflammation in RASFs.