DNA methylation regulates miR-140-5p and miR-146a expression in osteoarthritis

Aims: Previous studies have demonstrated that transcriptional silencing of miRNAs due to DNA hypermethylation is associated with different pathologies. It has also been reported that abnormal expression of miR-140-5p and miR-146a is linked to osteoarthritis (OA) progression. In this study, we investigated the role of DNA methylation on miR-140-5p and miR-146a expression in OA. Main methods: miR-140-5p and miR-146a expression was investigated by qRT-PCR. The methylation status of miR-140 and miR-146a regulatory regions was analyzed using qMSP and bisulfite sequencing analysis. SMAD-3 and NF-kB binding to miR-140 and miR-146a regulatory regions was assessed by ChIP assay and knockdown experiments. OA-related genes' expression was evaluated in 5-AzadC, miRNAs inhibitor and 5-AzadC/miRNAs inhibitor-treated cells. Key findings: Hypermethylation of specific CpG sites in miR-140 and miR-146a regulatory regions was associated with downregulation of miR-140-5p and miR-146a in OA chondrocytes and synoviocytes, respectively. 5-AzadC-induced miR-140-5p and miR-146a upregulation was observed in OA chondrocytes and synoviocytes. Moreover, we found decreased binding affinity of SMAD-3 and NF-kB transcription factors on the hypermethylated miR140-5p and miR-146a regulatory regions, respectively. Downregulation of MMP-13 and ADAMTS-5 in 5-AzadC-treated OA chondrocytes was prevented by miR-140-5p inhibitor transfection. Similarly, 5-AzadC-treated OA synoviocytes showed decreased expression of IRAK-1, IL1B and IL-6, which was reversed following 5-AzadC-/miR-146a inhibitor treatment. Significance: Our results strongly suggest the impact of DNA methylation on miR-140-5p and miR-146a suppression in OA chondrocytes and synoviocytes, contributing to OA pathogenesis.