MiR-206 Controls the Phenotypic Modulation of Pulmonary Arterial Smooth Muscle Cells Induced by Serum from Rats with Hepatopulmonary Syndrome by Regulating the Target Gene, Annexin A2

Abstract

Background: Hepatopulmonary syndrome (HPS) is a serious complication of advanced liver disease that is characterised by intrapulmonary vascular dilatation (IPVD) and arterial hypoxemia. Pulmonary vascular remodelling (PVR) is an important pathological feature of HPS, but the potential mechanisms underlying PVR remain undefined. Recent findings have established the essential role of changes in Annexin A2 (ANXA2) in controlling the phenotypic modulation of pulmonary artery smooth muscle cells (PASMCs) in PVR associated with HPS. However, the mechanism by which upstream signalling regulates ANXA2 is unclear. Methods: In the present study, computational analysis was used to predict which miRNA might target the 3´-untranslated region (3´-UTR) of the ANXA2 mRNA. Real-time PCR and western blotting were performed to study the level of correlation between ANXA2 and the differentiation marker with the predicted miRNAs in PASMCs stimulated with serum from normal rats or those with HPS. Functional analysis of the miRNA and a luciferase reporter assay were performed to demonstrate that the predicted miRNA suppressed ANXA2 expression by directly targeting the predicted 3´-UTR site of the ANXA2 mRNA. Results: Computational analysis predicted that miR-206 would target the 3´-UTR of ANXA2 mRNA. In HPS rat serum-stimulated PASMCs, the expression of miR-206 displayed an inverse correlation with ANXA2, while a positive correlation was observed with the phenotypic marker smooth muscle α-actin (SM α-actin). The miRNA functional analysis and luciferase reporter assay demonstrated that miR-206 effectively downregulated the expression of ANXA2 by binding to the 3´-UTR of the ANXA2 mRNA. Consistently, miR-206 effectively inhibited the HPS rat serum-induced phenotypic modulation and proliferation, while these effects were reversed in ANXA2-overexpressing PASMCs. Conclusion: This study demonstrates that miR-206 inhibits the HPS rat serum-induced phenotypic modulation and proliferation in PASMCs by down-regulating ANXA2 gene expression.