2022miR-216a、miR-301a调控BMPR2促进缺氧环境下肺动脉平滑肌细胞的增殖

目的: 探讨miR-216a、miR-301a对缺氧环境下人肺动脉平滑肌细胞（human pulmonary arterial smooth muscle cells,HPASMC）增殖的影响并研究其发生机制。方法: 采用CCK8法检测常氧和不同缺氧时间培养下HPASMC的增殖活力,qRT-PCR检测各组miR-216a、miR-301a和骨形成蛋白2型受体（bone morphogenetic protein receptor type 2,BMPR2）mRNA表达。CCK8和蛋白免疫印迹法分别检测转染了miR-216a或miR-301a抑制物后HPASMC的增殖能力及增殖细胞核抗原（proliferating cell nuclear antigen,PCNA）表达水平。双荧光素酶报告分别检测BMPR2与miR-216a、BMPR2与miR-301a之间的关系。CCK8和蛋白免疫印迹法分别检测同时转染miR-216a或miR-301a联合BMPR2过表达质粒后HPASMC的增殖能力及BMPR2、PCNA的表达水平。结果: 随着缺氧时间的延长,HPASMC的增殖活力和miR-216a表达增加,BMPR2 mRNA表达逐渐下降,miR-301a在缺氧24 h表达水平最高。下调miR-216a和miR-301a均显著抑制HPASMC的增殖活力及PCNA表达。双荧光素酶报告测定证实BMPR2分别是miR-216a和miR-301a的靶点。同时转染miR-216a或miR-301a联合BMPR2过表达质粒,BMPR2表达下降,细胞增殖活力及PCNA表达增加。结论: 缺氧环境下,miR-216a、miR-301a通过抑制BMPR2促进HPASMC增殖,引发肺动脉高压的形成。

miR-216a and miR-301a promote the proliferation of pulmonary arterial smooth muscle cells under hypoxia via regulating BMPR2

Objective: To investigate the effects of miR-216a and miR-301a on the proliferation of human pulmonary arterial smooth muscle cells （HPASMC） under hypoxia and to explore the potential mechanisms. Methods: The proliferation activity of HPASMC under normoxia and different hypoxia time was detected by CCK8 assay, and qRT-PCR was used to detect the expression of miR-216a, miR-301a and bone morphogenetic protein receptor type 2 （BMPR2） mRNA in each group. The proliferation ability and proliferating cell nuclear antigen （PCNA） expression levels of HPASMC transfected with anti-miR-216a or anti-miR-301a mimics were detected by CCK8 assay and Western blotting, respectively. The relationship between BMPR2 and miR 216a, BMPR2 and miR 301a were detected by dual luciferase reporter assay. CCK8 assay and Western blot were used to detect the proliferation ability of HPASMC and the expression levels of BMPR2 and PCNA after simultaneous transfection of miR-216a or miR-301a combined with BMPR2 overexpression plasmid, respectively. Results: With the prolongation of hypoxia time, the proliferation activity of HPASMC and the expression of miR-216a increased, while the expression of BMPR2 mRNA gradually decreased. The expression level of miR-301a was the highest in HPASMC exposed to hypoxia for 24 hours. Down-regulation of miR-216a and miR-301a significantly inhibited the proliferation activity and PCNA expression of HPASMC. Dual luciferase reporter assay confirmed that BMPR2 was a target of miR-216a and miR-301a, respectively. Simultaneously transfected with miR-216a or miR-301a combined with BMPR2 overexpression plasmid, the expression of BMPR2 decreased, and the cell proliferation activity and PCNA expression increased. Conclusion: miR-216a and miR-301a induce pulmonary arterial hypertension by promoting the proliferation of HPASMC under hypoxia via inhibiting the expression of BMPR2. ［Key words］miR-216a; miR-301a; pulmonary arterial hypertension; pulmonary arterial smooth muscle cells; bone morphogenetic protein receptor type 2