MicroRNA-138 Suppresses Osteoblastic Differentiation of Valvular Interstitial Cells in Degenerative Calcific Aortic Valve Disease

The aim of this study was to explore the function of miR-138 in the pathogenesis of degenerative calcific aortic valve disease (DCAVD). Aortic valve calcification tissue and normal tissue from DCAVD patients were collected to detect the expression of miR-138 by qRT-PCR, and immunohistochemical staining was performed to identify the phenotype of valve interstitial cells. QRT-PCR was performed to analyze the expression of miR-138, Runx2, MSX2, and ALP at day 7 after osteogenic differentiation. Alkaline phosphatase activity assay was performed at day 14 after osteogenic differentiation. Alizarin red staining was used to analyze the calcium nodule formation. TargetScan was used to predict potential targets of miR-138. QRT-PCR and Western blotting were perforined to analyze the expression of FOXC1 in valve interstitial cells (VICs). The aortic valve calcification was evaluated by quantitative analysis of the velocity in the aortic annulus and transvalvular pressure gradients. In this study, we demonstrated the role of miR-138 in VIC osteogenesis. QRT-PCR results revealed miR-138 was significantly down-regulated in calcified aortic valves compared with non-calcified valves. MiR-138 overexpression inhibited VIC osteogenic differentiation in vitro, while down-regulation of miR-138 enhanced the process. Target prediction analysis and dual-luciferase reporter assay confirmed FOXC1 was a direct target of miR-138. Further research found FOXC1 overexpression promoted VIC osteogenic differentiation. In addition, animal experiments validated indirectly miR-138 could suppress aortic valve calcification. Our findings suggest miR-138 could function as a new inhibitor of VIC osteogenic differentiation, which may act by targeting FOXC1 .