Background/Purpose:   TNF-α is a major cytokine implicated in rheumatoid arthritis (RA) and its expression has shown to be regulated at transcriptional and posttranscriptional levels. However, the impact of changes in microRNA (miRNA) expression on posttranslational processes involved in TNF-α signaling networks is not well-defined in RA. Here we evaluated the effect of miR-17 on TNF-α signaling pathway in human RA synovial fibroblasts (RASFs).

Methods:   MiR-17 expression was correlated in SFs/synovial tissue (ST) and serum from RA, osteoarthritis (OA), or healthy (NL) donors using qRT-PCR. Expression of miR-17 was also validated in the rat adjuvant-induced arthritis (AIA) model of RA. RNA sequencing was performed in RASFs transfected with pre-miR-17 or NC-pre-miR using an Ion Proton™ System. Effect of miR-17 on the expression of TNF-α signaling proteins (TRAF2, cIAP1, cIAP2, USP2, and PSMD13) at the basal level and in TNF-α (20ng/ml) stimulated RASFs was determined using Western immunoblotting. Immunoprecipitation (IP) and Western immunoblotting methods were used to determine the effect of miR-17 on the ubiquitination processes in RASFs. Culture supernatants from miR-17 overexpressing and TNF-α-stimulated RASFs were used to study the effect of miR-17 on IL-6, IL-8, production using cytokine array (Ray Biotech). Effect of miR-17 on the nuclear translocation of p-c-Jun and p-STAT3 was studied in TNF-α-stimulated RASFs. Statistical value of P<0.05 was considered significant.

Results:   We demonstrate that miR-17 expression was significantly low in the serum, SFs, and STs from RA patients as well as in the serum and joints of AIA rats. RNA sequencing analysis showed modulation of 664 genes by pre-miR-17 in human RASFs. Ingenuity pathway analysis of RNA sequencing data identified the ubiquitin proteasome system (UPS) in TNF-α signaling pathway as a primary target of miR-17. Western blot analysis confirmed the reduction of TRAF2, cIAP1, cIAP2, USP2, and PSMD13 expression by miR-17 in TNF-α-stimulated RASFs. IP assays showed that miR-17 restoration increased the K48-linked polyubiquitination of TRAF2, cIAP1, and cIAP2 in TNF-α-stimulated RASFs. Thus, destabilization of TRAF2 by miR-17 reduced the ability of TRAF2 to associate with cIAP2, thereby resulting in the downregulation of TNF-α-induced nuclear translocation of NF-ĸBp65, p-c-Jun, and p-STAT3 and the production of IL-6, IL-8, MMP-1, and MMP-13 in human RASFs.

Conclusion: This study provides a novel evidence for the role of miR-17 as a negative regulator of TNF-α signaling by modulating the protein ubiquitin processes in human RASFs.  

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/microrna-17-suppresses-tnf-%ce%b1-signaling-by-reducing-traf2-and-ciap2-association-in-rheumatoid-arthritis-synovial-fibroblasts/