MicroRNA 27B-3P: role in extracellular matrix regulation in osteoarthritis synovial fibroblasts

Abstract

Purpose: Synovial fibrosis is a key pathological event that contributes towards joint destruction, pain and stiffness associated with osteoarthritis (OA). Fibrosis is associated with the accumulation of excessive amount of extra-cellular matrix (ECM) resulting in tissue damage. Currently, endogenous mechanisms that play a key role in the initiation and progression of synovial fibrosis during OA are not well understood. We have previously shown that the expression of a small non-coding RNA called microRNA27b-3p (miR-27b-3p) is elevated in the synovial fluid of late-stage (Kellgren-Lawrence, KL score 3 & 4) compared to early-stage (KL score 1 & 2) radiographic knee OA patients. Furthermore, we also observed that miR-27b-3p is abundantly expressed in OA synovium (Y-H. Li, G. Tavallaee, et al. 2016). Since the role of miR-27b-3p in synovial pathology associated with OA has never been studied; we investigated if miR-27b-3p plays any role in ECM regulation in the OA synovium. Methods: Synovial tissues were obtained from OA patients undergoing total knee replacement with informed written consent. Primary OA synovial fibroblasts (major cell types present in the synovium) were isolated, cultured and transfected with miR-27b-3p mimic/inhibitor and subjected to qPCR arrays specific for ECM markers. Results obtained from arrays were further validated using quantitative-PCR analysis. For in vivo studies, mice were also subjected to DMM model of OA to determine the expression of miR-27b-3p during the time course of OA development in knee joint using in situ hybridization (ISH). Results: ECM-specific qPCR-arrays followed by validation of responsive genes by quantitative-PCR analysis revealed that miR-27b-3p mimic treatment resulted in a significant upregulation of key ECM genes including pro-collagen type-I (COL1A1), A disintegrin and metalloproteinase with thrombospondin motifs 8 (ADAMTS-8), Thrombospondin 1 (THBS1), fibronectin1 (FN1), Collagen 5 A 1 (COL5A1) and Collagen 14 A 1 (COL14A1) in OA Synovial fibroblasts. Furthermore, miR-27b-3p mimic resulted in a marked increase in type-I collagen protein expression; this effect was inhibited by miR-27b-3p inhibitor. Overall, these results suggest that miR-27b-3p may play a key role in the regulation of key ECM components in OA synovial fibroblasts. Next, we employed “Watson Discovery tool” to identify direct targets of miR-27b-3p to further elucidate its signalling pathway. This tool identified a panel of 11 genes including peroxisome proliferator-activated receptor gamma (PPARγ) as direct targets for miR-27b-3p. Indeed, we confirmed that miR-27b-3p mimic treatment results in a marked suppression of PPARγ expression, while inhibition of miR-27b-3p upregulates PPARγ, suggesting that miR-27b-3p may impart its ECM regulatory effect through modulation of PPARγ. Furthermore, ISH of DMM-induced OA mice knee joints further show that miR-27b-3p expression is prominent in the synovial lining and fibrocartilage compared to sham control mice in vivo. Conclusions: Our results thus far suggest that, in OA synovial fibroblasts, miR-27b-3p is involved in the regulation of key ECM components. Our data also suggest that miR-27b-3p may signal via negative regulation of PPARγ. Our ongoing studies are confirming if miR-27b-3p signalling via PPARγ controls ECM regulation in OA synovial fibroblasts. Our in vivo studies show that miR-27b-3p expression is elevated in the synovium during OA. To comprehensively investigate the endogenous role of miR-27b-3p in synovial pathology, we have intra-articularly injected mouse knee joints with the in-vivo grade mimic of miR-27b-3p to determine any changes in the synovium upon miR-27b-3p treatment in vivo.