Expression And Molecular Mechanism Of MicroRNA-206 In Rat Hypoxia-induced Pulmonary Hypertension

Background:Pulmonary hypertension (PH), a serious disease characterized by high blood pressure in the lungs and enhanced proliferation of pulmonary artery smooth muscle cells (PASMCs), is the most common clinical manifestation of chronic obstructive pulmonary disease (COPD). What that triggering the development of this syndrome is poorly understood but of far-reaching significance. MiRs, a class of～23 nt, noncoding single-stranded RNAs that regulate gene function by identifying the 3' Untranslated Regions (UTRs) of targeted gene, leading to a significant negative regulation of relevant mRNAs to be translated into proteins. MiR-1 family has been proven to be muscle-specific miRs and played critical roles in myogenesis and growth. It was not clear whether the MiR-1 family is implicated in the etiology of hypoxia-induced PH.Objective:The present study was aimed to investigate the expression and role of muscle-specific microRNA-206 (miR-206) during hypoxia-induced PH.Methods:Rats first received short-term and sustained hypoxic exposure in vivo. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the levels of miR-206 in pulmonary tissue and serum, and the levels of HIF-1αand Fhl-1 in pulmonary tissue. Pre-miRNA-206 and small-interfering RNA targeting Fhl-1 were transfected into pulmonary artery smooth muscle cells (PASMCs) respectively by transient transfection and the cells were cultured in hypoxia condition. Subsequently, expression of miR-206, HIF-1αmRNA and Fhl-1mRNA was evaluated by RT-qPCR, protein expression of Fhl-1, HIF-1αand Ki-67 was further determined by Western blot, immunohistochemistry and immunofluorescence. Flow cytometry analysis was used to examine the cell cycle of PASMCs. HIF-1αgene was screened for miRNA response elements (MREs).Results:In comparison with the controls, significantly down-regulation of miR-206 in lung tissue was observed after 1 day of hypoxia, and the level maintained a consistent low level in the prolonged hypoxic period. Serum level of miR-206 was higher after a short-time hypoxic exposure, and reached a peak at 4th day, after that, there was a decreasing tendency over hypoxia treatment time. The levels of Fhl-1 were up-regulated in lung tissue after short-term and prolonged hypoxic treatment. Similar change of miR-206 and Fhl-1 levels was further observed in PASMCs subjected to 1 day of hypoxia. Transfection of Pre-miR miR-206, followed by hypoxia exposure, led to over expression of miR-206, increased expression of Fhl-1, promoted S phase cells in the cell cycle and proliferation in PASMCs. Down-regulation of Fhl-1 expression by siRNA led to increased G1 phase cells and reduced proliferation, however, it did not affect the expression of miR-206. A binding site of miR-206 was located in 3'UTR region of HIF-1αgene.Conclusion:MiR-206 is a novel biomarker for early rat pulmonary hypertension and'exogenous'transfected miR-206 has a saturation effect on HIF-1α/Fhl-1 pathway in the process..