Effect of MicroRNA-26a Targeting mCTGF on Calcification of Vascular Smooth Muscle Cells Through Regulating Osteoprotegerin/Receptor Activator of NF-kB/Receptor Activator of Nuclear Factor Kappa-B Ligand Signaling Pathway

Abstract

To explore the effect of microRNA-26a targeting mCTGF on inhibiting the calcification of vascular smooth muscle cells (VSMCs) through OPG/RANK/RANKL signaling pathway. VSMCs were isolated and cultured by adherent method. The morphology of VSMCs was observed under microscope and the surface antigen was identified by flow cytometry. The calcification of VSMCs was detected using alizarin red S staining and oil red O staining. microRNA-26a level in supernatant of cultured VSMCs was detected by ultracentrifugation method. The binding site between microRNA-26a and 3′-UTR of mCTGF was predicted by bioinformatics software; VSMCs were induced to be calcified and verified by the luciferase reporter gene assay. The mRNA and protein expressions of OPG/RANK/RANKL signaling pathway were measured by qRT-PCR and Western blotting. Normal VSMCs were assigned into normal group (normal VSMCs were treated with the exosomes secreted by normal VSMCs), CKD group (normal VSMCs were treated with the exosomes secreted by VSMCs in mice with chronic kidney disease (CKD)) and CKD/overexpressed mCTGF group (normal VSMCs were treated with the exosomes secreted by VSMCs in CKD mice and meanwhile treated with overexpressed mCTGF) followed by analysis of OPG/RANK/RANKL signaling level by qRT-PCR and ALP activity. VSMCs grew against the wall and showed a long fusiform shape. Calcification and adipogenesis of VSMCs was found under different induction conditions. microRNA-26a level in the supernatant of VSMCs in CKD group was significantly higher than normal group; after microRNA-26a bound to of mCTGF, mCTGF level in the CKD/overexpressed mCTGF group was significantly lower. The expression of OPG/RANK/RANKL signaling pathway was significantly decreased in the CKD/overexpressed mCTGF group. Meanwhile, OPG/RANK/RANKL signaling protein was increased significantly after treatment of exosomes secreted by VSMCs in CKD mice. mCTGF is the target gene of microRNA-26a and microRNA-26a can target mCTGF to regulate exosomes secretion by VSMCs and OPG/RANK/RANKL signaling to inhibit the calcification of VSMCs.