Identification of synovial fluid MIRNA signature in knee osteoarthritis: differentiating early and late knee osteoarthritis

Abstract

Purpose: Osteoarthritis (OA) is a degenerative joint disease characterized by the progressive breakdown of articular cartilage. While cartilage degradation remains the hallmark of OA, it is clear that all joint tissues contribute to the pathological process, however, the molecular mechanisms that drive these degenerative events remain poorly understood. This highlights the critical need to perform parallel molecular studies on articular cartilage and other OA-affected joint tissues, such as the synovium (SYN) and underlying subchondral bone (SCB). Intriguingly, an increasing number of miRNAs are being identified as novel regulators of OA disease initiation and progression, making them exciting candidates for therapeutic targets and diagnostic biomarkers. The purpose of this study is to investigate previously published candidate miRNAs, known to be dysregulated in human OA, and determine their role in extra-cartilaginous OA joint tissues. Methods: OA was induced in 10e12 week old male wild type mice by bilateral surgical destabilization of the medial meniscus (DMM). RNA from SCB from DMM and sham-operated mice was isolated by laser microdissection at 1 and 6 weeks post-surgery. RNA was also extracted from the SYN of the same mice. miRNA expression profiling of SCB and SYN was performed using Agilent miRNA microarrays. Histological measurements examining the severity of OA in the contralateral joint (including: SCB sclerosis; osteophyte size and maturity; anterior and posterior synovitis -panus presence and bone erosion, sub-synovial inflammatory cell infiltration, synoviocyte hyperplasia, and exudate) were scored by one observer blinded to surgical intervention. Results: There was no difference in SCB sclerosis between surgeries at 1 week but DMM > sham at 6 weeks (p ¼ 0.003), and DMM-6wk > DMM-1wk (p ¼ 0.04). There was no osteophyte development at 1 week but at 6 weeks they had formed in DMM only, being larger (p ¼ 0.002) and more mature (p ¼ 0.001) than sham. Anterior and posterior synovitis decreased with post-operative time after sham and DMM (1wk > 6wk, p < 0.01 for all comparisons). There was no difference between surgeries in the individual synovitis parameters in the anterior aspect of the joint at either time, other than more panus in DMM at 1 week (p ¼ 0.049). However, more severe joint inflammation in DMM compared with sham was evident by significantly higher synovitis scores in the posterior region of the joint (distant from the surgical incision) at both 1 and 6 weeks (p ¼ 0.01 and 0.03, respectively). miRNA expression analysis revealed 584 miRNAs to be differentially expressed between SYN and SCB samples (adj.p.value < 0.05). Moreover, 384 and 164 miRNAs were dysregulated between 1 and 6 week time points (adj.p.value < 0.05) in SYN and SCB, respectively. However, there were no changes in miRNA expression between DMM and sham mice at both 1 and 6 weeks post-surgery in either the SCB or SYN. Additionally, promising candidate miRNAs previously identified in human OA cartilage (for example, miR-140, miR-...

microRNA‑mediated GAS1 downregulation promotes the proliferation of synovial fibroblasts by PI3K‑Akt signaling in osteoarthritis

“…The mechanism by which IL-1β induces the downregulation of GAS1 remains unclear; therefore, it was hypothesized that endogenous small miRs, induced by pro-inflammatory factors, may modulate GAS1 expression by directly targeting the 3′UTR of GAS1 mRNA. In particular, three miRs, miR-34a-5p, miR-203a-3p and miR-181a-5p, were found to be overexpressed in OASFs according to a number of previously published reports (21,23) and were capable of binding to the 3′UTR of GAS1 mRNA, according to the Targetscan 7.2 predictions. Therefore, the expression of these three miRs in OASFs and non-OASFs were measured using RT-qPCR.…”

“…It was speculated that miRs could be important mediators of GAS1 expression due to their reported regulatory functions by post-transcriptionally binding to specific sequences of target gene 3′UTRs. Following investigations into miRNA-gene matching using a combination of miRNA expression profile data of OA and non-OASFs from other studies (21,23), three miRNAs, miR-34a-5p, miR-203a-3p and miR-181a-5p, which are found to be upregulated in OASFs and have potential binding sites in the GAS1 3′UTR as predicted by Targetscan 7.2, were selected for further analysis in the present study. miR-34a-5p and miR-181a-5p, but not miR-203a-3p, were found to be significantly overexpressed with a >7-fold change, in OASFs and IL-1β-treated non-OASFs, by RT-qPCR.…”

“…By entering GAS1 in the ‘Gene symbol’, a list of miRNA families whose seed regions match the 3′untranslated region (3′UTR) of GAS1 were obtained. In total, 18 miRNA families and corresponding binding sites were listed as ‘broadly conserved among vertebrates.’ Following a literature search, two previous studies were found showing differentially expressed miRNAs between OA and non-OA synovial tissues, synovial fluids or synovial fibroblasts (21,23). By pooling the data obtained from the predicted miRNA list and these two studies, three miRNA candidates potentially regulating GAS1 expression in OASFs were shortlisted.…”

“…Indeed, numerous studies have reported that miR-146a, miR-155, miR-124a and miR-126 are widely implicated in inflammation or hyperplasia in synovial tissues and OA pathogenesis (18–20). Considering miRNA sequencing data (21–23) and Targetscan 7.2 prediction results, it was hypothesized that potential miRNA candidates that can directly regulate GAS1 expression may exist in OASFs.…”

Altered expression of microRNA-98 in IL-1β-induced cartilage degradation and its role in chondrocyte apoptosis

“…Numerous etiological factors are implicated in the pathogenesis of OA; however, cartilage destruction appears to be due to chondrocyte apoptosis (1). It has previously been suggested that microRNAs (miRNAs/miR) are involved in the pathogenesis of OA (2). miRNAs are single-stranded, non-coding small RNAs that negatively regulate the expression of target genes in a post-transcriptional manner; >700 miRNAs have been identified to serve a role in the regulation of cellular processes, including proliferation, apoptosis and differentiation (3,4).…”