Role of TRPV4 in methylation of the CpG sites on miR146a promoter. Role of TRPV4 in methylation of the CpG sites on miR146a promoter

Epigenetic modifications, including the role of miRs in various diseases, are key regulators of gene expression. In this study we aimed to explore whether epigenetic alterations could play a role in the TRPV4-mediated regulation of miR-146a. Here we report that there is no significant differences in the methylation status of the examined 10 CpG sites on miR146a promoter between WT and TRPV4 KO cells under LPS treatment, indicating that the regulation of miR-146a by TRPV4 may not be mediated by methylation changes at these specific sites. Overall design: To investigate the role of TRPV4 on methylation of CpG sites on miR146a promoter, we isolated Thioglycolate induced peritoneal macrophages from WT and TRPV4KO mice. Cells were either left untreated or treated with eLPS(100ng/ml). Cell pellets were lysed and bisulphite modification was performed. Bisulphite modified DNA was then amplified using multiplex or simplex PCRs. Libraries were next prepared and sequenced on the Ion S5TM sequencer using an Ion 530TM sequencing chip. FASTQ files from the Ion Torrent S5 server were aligned to a local reference Methylation levels were calculated.