miR-146-5p restrains calcification of vascular smooth muscle cells by suppressing TRAF6.

Abstract

Vascular calcification is a prominent manifestation of advanced atherosclerosis. Tumor necrosis factor-receptor-associated factors (TRAFs) were reported to participate in atherosclerosis development. In this study, the role and mechanism of TRAF6 in vascular calcification were explored. To induce the vascular calcification, oxidized low-density lipoprotein (Ox-LDL) was applied to treat vascular smooth muscle cells (VSMCs). TRAF6 protein expression in VSMCs was assessed by western blotting. Osteogenic differentiation of VSMCs was assessed by alkaline phosphatase activity analysis. Mineral deposition in VSMCs was evaluated by von Kossa staining. VSMC proliferation, migration, apoptosis, inflammation, and reactive oxygen species (ROS) generation were detected using cell counting kit-8, Transwell, flow cytometry, reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), and dichlorodihydrofluorescein diacetate staining, respectively. Luciferase reporter assay was utilized to identify the binding relationship between miR-146-5p and TRAF6 in VSMCs. We found that Ox-LDL administration induced the calcification of VSMCs and elevated the TRAF6 level. TRAF6 knockdown restrained VSMC calcification, proliferation, migration, inflammation, and ROS generation caused by Ox-LDL. Mechanically, TRAF6 was targeted by miR-146-5p in VSMCs. Furthermore, TRAF6 overexpression offset the inhibitory effects of miR-146-5p upregulation on vascular calcification in VSMCs under the Ox-LDL condition. Overall, miR-146-5p restrains the calcification of VSMCs by suppressing TRAF6.