Hypermethylation of miR-181b in monocytes is associated with coronary artery disease and promotes M1 polarized phenotype via PIAS1-KLF4 axis.

Background: Dysregulated microRNAs are involved in the macrophage polarization and atherosclerotic development. Apart from microRNAs, alteration in DNA methylation is considered as one of the most frequent epigenetic changes. The purpose of the research is to investigate the altered methylation status of miR-181b in the circulating monocytes from patients with coronary artery disease (CAD) and explore the underlying mechanisms. Methods: We examined the methylation status of miR-181b in purified circulating monocytes from patients with CAD and healthy controls. We then transfected monocytes with miR-181b mimics and determined the role of miR-181b on the phenotypic switch of macrophages and inflammatory response. DNA methylation levels determined by MethyLight PCR and pyrosequencing at the promoter of rniR-181b significantly increased in CAD patients. Based on TargetScan database, we identified MASI as the target gene of miR-181b and explored the interaction of miR-181b and PIAS1 by Dual-Luciferase assay, quantitative PCR and immunoblots. We also investigated the role of miR-181b and PIAS1 on macrophage polarization and inflammation. Results: Hypermethylation at the promoter of miR-181b directly contributed to the decrease of miR-181b activity and expression. Overexpression of miR-181b reduced M1 polarization and facilitated M2 polarization determined by quantitative PCR. While knockdown of PIAS1 induced KLF4 degradation and SUMOylation in monocytes, miR-181b mimics reverse the KLF4 SUMOylation via suppression of PIAS1. Moreover, KLF4 SUMOylation by PIAS1 reversed M1 polarization induced by depletion of miR-181b in monocytes. Conclusions: Hypermethylation of miR-181b induces M1 polarization and promotes atherosclerosis through activation of PIAS1 and KLF4 SUMOylation in macrophages.