Research on the Expression and Mechanism of miR-204 in Pulmonary Hypertension Model Rats
Objective:To explore the role of miR-204 in pulmonary arterial hypertension(PAH)model rats and its related molecu-lar mechanism.Methods:PAH model rats were constructed by one-time intraperitoneal injection of monocrotaline(MCT,60 mg/kg)so-lution.PAH rats were divided into PAH group,NC group and miR-204 group,with 10 rats in each group.Another 15 healthy SD rats were selected as control group.The lesions of lung and right ventricle tissues were detected by HE staining,and the expression level ofα-SMA protein in lung tissues was detected by immunohistochemical staining.The relationship between miR-204 and Runx2 was ana-lyzed using dual luciferase reporter genes.Human pulmonary arterial smooth muscle cells(PASMCs)were divided into control group,hypoxia group,mimics NC group,miR-204 mimics group,pcDNA3.1 group and Runx2 group.The proliferation of PASMCs cell was detected by CCK-8 method and EdU method,the cell cycle distribution was detected by flow cytometry,and the migration of PASMCs cell was detected by Transwell assay.The mRNA expression levels of miR-204 and Runx2 in lung tissue and PASMCs cells were detected by qRT-PCR.The protein expression levels of Runx2,α-SMA,CDKN4,Cyclin E and Cyclin D1 in lung tissue or PASMCs cells were detected by Western blot.Results:Compared with the control group,the expression level of miR-204 in lung tissue of rats in PAH group was decreased(P＜0.05),the expression levels of Runx2 and α-SMA protein were increased(P＜0.05),the pulmonary artery wall was thickened,lumen was narrow,and cardiomyocyte hypertrophy was observed.Compared with the NC group,the expression level of miR-204 in the lung tissues of rats in miR-204 group was increased(P＜0.05),and the expression levels of Runx2 and α-SMA protein were decreased(P＜0.05),and the thickening of pulmonary artery wall and cardiomyocyte hypertrophy of rats were significantly im-proved.miR-204 has a targeted binding relationship with Runx2.Compared with the control group,the expression level of miR-204 and CDKN4 protein,and the distribution ratio of G0/G1 phase were decreased in hypoxia group(P＜0.05),and the expression levels of Runx2 mRNA and protein,Cyclin E and Cyclin D1 protein were increased(P＜0.05).The cell proliferation level and migration number were in-creased(P＜0.05).Compared with the mimics NC group,the expression level of miR-204 and CDKN4 protein,and the distribution ratio of G0/G1 phase were increased in miR-204 mimics group(P＜0.05),and the expression levels of Runx2 mRNA and protein,Cyclin E and Cyclin D1 protein were decreased(P＜0.05).The cell proliferation level and migration number were decreased(P＜0.05).Compared with the pcDNA3.1 group,the expression level of miR-204 and CDKN4 protein,and the distribution ratio of G0/G1 phase were decreased in Runx2 group(P＜0.05),and the expression levels of Runx2 mRNA and protein,Cyclin E and Cyclin D1 protein were increased(P＜0.05).The cell proliferation level and migration number were increased(P＜0.05).Conclusion:miR-204 is underexpressed in PAH model rats,and overexpression of miR-204 can inhibit the proliferation and migration of PASMCs cells and promote cell division arrest in the G0/G1 phase,which may be related to the targeted inhibition of Runx2 protein expression.
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