DNA methylation regulates MiR-140 expression in primary osteoarthritic chondrocytes by altering SMAD-3 binding affinity

Abstract

Purpose: Osteoarthritis (OA) is a whole-joint disorder that is characterized by the loss of articular cartilage of synovial joints. Abnormal expression of miRNAs, through changes of DNA methylation status of their regulatory regions has been suggested to contribute to pathogenic alterations of an OA joint. MiR-140 is specifically expressed in cartilage and its reduced expression has been associated with abnormal chondrocytes’ metabolism in OA. The aim of the present study was to investigate the role of DNA methylation on miR-140 expression in osteoarthritic chondrocytes. Methods: Articular osteoarthritic and normal cartilage were obtained from 16 patients with primary osteoarthritis and 9 individuals with no history of joint disease, respectively. MiR-140 mRNA expression levels were investigated using real-time PCR. The methylation status of miR-140 regulatory region was analysed using quantitative methylation-specific PCR (qMSP) and bisulfite DNA sequencing assay. The effect of 5’-Aza-2-deoxycytidine (5-AzadC) on miR-140 expression was investigated and SMAD-3 binding to miR-140 regulatory region was assessed by chromatin immunoprecipitation (ChΙP). Statistical analysis was performed using the SPSS software 24. MiR-140 relative expression was assessed using Mann-Whitney. Spearman’s rank correlation coefficient was used to analyze correlative relationships. For all comparisons, p values < 0.05 were considered statistically significant and data were expressed as the mean ± standard error of the mean. Results: We observed that miR-140 expression levels were significantly reduced in OA compared with normal chondrocytes. Downregulation of miR-140 was correlated with increased percentage of methylation of miR-140 regulatory region in OA chondrocytes. Bisulfite DNA sequencing of miR-140 regulatory region revealed 6 CpG sites located near SMAD-3 binding sites to be highly methylated in OA compared to normal chondrocytes. We also observed a weaker binding affinity of SMAD-3 in OA chondrocytes compared to normal, whose miR-140 regulatory region was hypermethylated. Treatment of OA chondrocytes with 5-AzadC resulted in significant upregulation of miR-140 compared to untreated, which was associated with demethylation of miR-140 regulatory region and stronger binding of SMAD-3 in this region. Conclusions: We provide novel findings that DNA methylation regulates miR-140 expression in OA chondrocytes, by changing SMAD-3 binding affinity to miR-140 regulatory region. Our results provide strong evidence that abnormal expression of miRNAs observed in OA is regulated by epigenetics, such as DNA methylation, suggesting a new strategy for treating OA patients, as epigenetic mechanisms are reversible processes.