Wang Zhonghua, Li Chunlei, Sun Xinyong, Li Zhuqin, Li Jia, Wang Lanfeng, Sun Yanming
Department of Cardiology, The First Affiliated Hospital of Harbin Medical University, Harbin, China.
Cardiovasc Diagn Ther. 2020 Aug;10(4):738-751. doi: 10.21037/cdt-20-407.
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.
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 miR-181b significantly increased in CAD patients. Based on TargetScan database, we identified PIAS1 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.
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.
Hypermethylation of miR-181b induces M1 polarization and promotes atherosclerosis through activation of PIAS1 and KLF4 SUMOylation in macrophages.
微小RNA失调参与巨噬细胞极化和动脉粥样硬化发展。除微小RNA外，DNA甲基化改变被认为是最常见的表观遗传变化之一。本研究旨在调查冠心病（CAD）患者循环单核细胞中miR-181b的甲基化状态改变，并探索其潜在机制。
我们检测了CAD患者和健康对照者纯化循环单核细胞中miR-181b的甲基化状态。然后用miR-181b模拟物转染单核细胞，并确定miR-181b对巨噬细胞表型转换和炎症反应的作用。通过甲基化荧光定量PCR和焦磷酸测序测定，CAD患者中miR-181b启动子处的DNA甲基化水平显著升高。基于TargetScan数据库，我们鉴定PIAS1为miR-181b的靶基因，并通过双荧光素酶测定、定量PCR和免疫印迹探索miR-181b与PIAS1的相互作用。我们还研究了miR-181b和PIAS1对巨噬细胞极化和炎症的作用。
miR-181b启动子处的高甲基化直接导致miR-181b活性和表达降低。通过定量PCR测定，miR-181b过表达减少M1极化并促进M2极化。虽然PIAS1的敲低诱导单核细胞中KLF4降解和SUMO化，但miR-181b模拟物通过抑制PIAS1逆转KLF4 SUMO化。此外，PIAS1介导的KLF4 SUMO化逆转了单核细胞中miR-181b缺失诱导的M1极化。
miR-181b的高甲基化通过激活巨噬细胞中的PIAS1和KLF4 SUMO化诱导M1极化并促进动脉粥样硬化。
