Background/Purpose: Synovial fibroblasts play crucial roles in the pathogenesis of rheumatoid arthritis (RA). They secrete proteinases and proinflammatory cytokines to damage the synovial tissues in the RA joints. Their functions are regulated by a number of molecules including microRNAs (miRNAs). MiRNAs are endogenous small noncoding RNAs, and suppress expression of target genes by binding to 3’- untranslated region (UTR) of the corresponding messenger RNAs. The present study was conducted to identify miRNAs to regulate expression of matrix metalloproteinase-3 (MMP-3) and interleukin-6 (IL-6), which are crucial in the pathogenesis of RA.

Methods: Nine hundred and twenty-four inhibitors of human miRNAs were transfected individually into synovial fibroblast cell lines developed from the RA synovial tissues. The transfected cells were cultured in the presence or absence of 1 ng/ml TNF-α for 24-72 hours. The expression levels of MMP-3 and IL-6 were quantified with enzyme-linked immunosorbent assay. The expression level of ets variant 6 (ETV6) was determined with Western blotting and quantitative polymerase chain reaction analysis. MiRNA array analysis was conducted for evaluation of the miRNA expression levels in the synovial fibroblasts with or without the TNF-α stimulation.

Results: Functional screening of 924 miRNAs using miRNA inhibitor library identified 14 miRNAs to regulate expression of MMP-3 in synovial fibroblasts. Out of these miRNAs, miR-103, miR-129-5p, miR-135a and miR-193b have seed sequence that match with 3’ UTR of ETV6, which is a transcriptional repressor of MMP-3. The effects of the four miRNA inhibitors on MMP-3 expression were validated with independent experiments. Inhibitors of miR-103 and miR-129-5p suppressed the expression level of IL-6. The four miRNA inhibitors increased the protein expression of ETV6 without upregulating the mRNA level. MiRNA array analysis revealed that the expression levels of miR-103, miR-129-5p and miR-135a in synovial fibroblasts were increased when they were stimulated with TNF-α.

Conclusion: Functional screening of miRNAs using the inhibitor library identified miR-103, miR-129-5p, miR-135a and miR-193b, which target ETV6, as regulators of MMP-3 and IL-6 expression in RA synovial fibroblasts. These miRNAs could be novel drug targets in RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/functional-screening-of-micrornas-using-the-inhibitor-library-identified-micrornas-to-regulate-expression-of-mmp-3-and-il-6-in-rheumatoid-arthritis-synovial-fibroblasts/