Downregulation of microRNA-637 Increases Risk of Hypoxia-Induced Pulmonary Hypertension by Modulating Expression of Cyclin Dependent Kinase 6 (CDK6) in Pulmonary Smooth Muscle Cells
Hai-yan Sang, Ying-li Jin, Wen-qi Zhang, Li-bo Chen
Department of Cardiology, China-Japan Union Hospital of Jilin University, Changchun, Jilin, China (mainland)
Med Sci Monit 2016; 22:4066-4072
The objective of this study was to investigate the molecular mechanism by which miR-637 interferes with the expression of CDK6, which contributes to the development of pulmonary hypertension (PH) with chronic obstructive pulmonary disease (COPD).
MATERIAL AND METHODS: We used an online miRNA database to identify CDK6 as a virtual target of miR-637, and validated the hypothesis using luciferase assay. Furthermore, we transfected SMCs with miR-637 mimics and inhibitor, and expression of CDK6 was determined using Western blot and real-time PCR.
RESULTS: In this study, we identified CDK6 as a target of miR-637 in smooth muscle cells (SMCs), and determined the expression of miR-637 in SMCs from PH patients with COPD and normal controls. We also identified the exact miR-637 binding site in the 3’UTR of CDK6 by using a luciferase reporter system. The mRNA and protein expression levels of CDK6 in SMCs from PH patients with COPD were clearly upregulated compared with the normal controls. Cells exposed to hypoxia also showed notably increased CKD6 mRNA and protein expression levels, and when treated with miR-637 or CDK6 siRNA, this increase in CKD6 expression was clearly attenuated. Additionally, cell viability and cell cycle analysis showed that hypoxia markedly increased viability of SMCs by causing an accumulation in S phase, which was relieved by the introduction of miR-637 or CDK6 siRNA.
CONCLUSIONS: Our study proved that the CDK6 gene is a target of miR-637, and demonstrated the regulatory association between miR-637 and CDK6, suggesting a possible therapeutic target for PH, especially in patients with COPD.
Keywords: Case-Control Studies, 3' Untranslated Regions, Cell Cycle Checkpoints - genetics, Cell Line, Tumor, Cell Proliferation - physiology, Cyclin-Dependent Kinase 6 - genetics, Down-Regulation, Humans, Hypertension, Pulmonary - metabolism, Hypoxia - metabolism, MicroRNAs - metabolism, Myocytes, Smooth Muscle - physiology, primary cell culture, RNA, Messenger - metabolism, RNA, Small Interfering