Bioinformatic Analysis Reveals Association of MIR-140-5P and MIR-146A Target Genes with Osteoarthritis-Related Processes

Purpose: Osteoarthritis (OA) is the most common degenerative joint disease and its incidence is steadily increasing affecting almost 250 million people worldwide. Although OA has long been considered as a non-inflammatory disease, it is now well-recognized that age-related inflammation and obesity-induced inflammation contribute to the inflammatory network seen within an OA joint. MiR-140-5p and miR-146a are deregulated in OA chondrocytes and is well-known that control OA-related processes including inflammation and chondrogenesis. In this study we performed bioinformatic and functional analysis to identify the processes and pathways regulated by OA-related target genes of miR-140-5p and miR-146a. Methods: MiR-140-5p and miR-146a target genes associated with OA was selected from 4 databases as well as from Open Targets and the miRNA-mRNA network was visualized using Cytoscape. Protein-protein interaction (PPI) network was constructed from STRING tool and MCODE plug-in was applied to identify significant modules. Gene ontology (GO) and pathway enrichment analysis were performed using Metascape tool. Articular osteoarthritic and normal cartilage were obtained from 25 patients with primary osteoarthritis and 12 individuals with no history of joint disease, respectively. TLR4 and TRAF-6 expression levels were investigated using quantitative real-time PCR (qRT-PCR) and western blot. OA cultured chondrocytes were treated with miR-140 and/or miR-146a mimic as well as with miR-140 and/or miR-146a inhibitors and the expression levels of TLR4, TRAF-6, NF-kB, IL-1β, IL-6, and TNF-a, were evaluated using qRT-PCR, western blot or ELISA assay. Results: 35 miR-140-5p and 14 of miR-146a target genes were common in 3 out of 4 databases and in Open Targets and were related to OA. PPI network analysis revealed that TLR4 was the only gene regulated by both miRNAs and had a high connectivity degree with other target genes. ΜCODE clustering analysis revealed 3 sub-network modules and TLR4 was located in the sub-network with the highest MCODE score. GO enrichment analysis revealed that MCODEs-related genes were involved in various OA related-biological processes, including regulation of cellular differentiation and signal transduction, ossification, osteoblast differentiation and inflammatory responses. Pathway enrichment analysis showed that the above genes were significantly enriched in 7 signaling pathways, some of which are associated with OA pathogenesis, as MAPK, TLRs, PI3K-Akt and NF-κB. TLR4 signaling pathway was found to be over-activated in OA chondrocytes, as we observed increased expression of TLR4 and TRAF6 in OA chondrocytes compared with normal. Overexpression of miR-140-5p or miR-146a and combined miR-140-5p/miR-146a overexpression in OA chondrocytes demonstrated that combined treatment had the strongest negative effect on TLR4 expression. Moreover, simultaneous overexpression of miR-140-5p and miR-146a resulted in the highest reduction of NF-κΒ phosphorylation levels, as well as IL-1b, IL-6 and TNFa expression levels in OA chondrocytes as compared to the reductions observed when either miR-140-5b or miR-146a was overexpressed. Conclusions: Bioinformatic analysis revealed that combinatory treatment using miR-140-5p and miR-146a mimics might be an effective strategy to block inflammation-mediated catabolism and other processes implicated in OA cartilage destruction. Mir-140-5p and miR-146a overexpression had a strong protective effect against inflammatory mediators’ production in OA chondrocytes through targeting the TLR4/NF-κB signaling. Simultaneous overexpression of miR-140-5p and miR-146a could be a promising new mean for future preclinical and clinical studies aiming at controlling OA onset and/or progression.