MiR-31a-5p通过靶向HIF-1α促细胞凋亡加重心肌缺血损伤  

MiR-31a-5p aggravates apoptosis in myocardial ischemia by targeting HIF-1α

作　　者：卢孔利 李雪晴 杜凌 薛松[1] 连锋[1] LU Kongli;LI Xueqing;DU Ling;XUE Song;LIAN Feng(Department of Cardiovascular Surgery,Renji Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai,200127,P.R.China;Department of Central Laboratory,Renji Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai,200127,P.R.China)

机构地区：[1]上海交通大学医学院附属仁济医院心血管外科,上海200127 [2]上海交通大学医学院附属仁济医院中心实验室,上海200127

出　　处：《中国胸心血管外科临床杂志》2024年第5期782-790,共9页Chinese Journal of Clinical Thoracic and Cardiovascular Surgery

基　　金：国家自然科学基金(81873461)。

摘　　要：目的 探究miR-31a-5p在心肌梗死(myocardial infarction,MI)小鼠中的表达及其潜在的作用机制。 方法 从基因表达数据库中下载数据集,并使用生物信息学方法筛选出miR-31a-5p及其预测靶基因缺氧诱导因子-1α(hypoxia-inducible factor-1α,HIF-1α)。通过结扎C57BL/6J雄性小鼠冠状动脉左前降支建立MI模型。小鼠随机分为假手术组(n=6)、模型组(n=6)。采用氯化钴(CoCl_(2))处理H9c2细胞诱导体外缺氧细胞模型,分为对照组、模型组、NC组、miR-31a-5p mimic组、miR-31a-5p inhibitor组;通过Masson染色并分析心肌组织纤维化程度;通过qRT-PCR检测小鼠心肌组织、H9c2细胞中miR-31a-5p和HIF-1α mRNA的表达水平;使用Western blotting分别检测小鼠心肌组织凋亡蛋白:B细胞淋巴瘤2(B-cell lymphoma 2,Bcl-2)、半胱氨酸蛋白酶-3剪切体(cleaved-caspase 3)以及H9c2细胞中HIF-1α和凋亡蛋白的表达水平。采用双荧光素酶报告基因测定验证miR-31a-5p与HIF-1α的靶向关系。 结果 Masson染色结果表明MI小鼠纤维化程度显著升高(P<0.000 1);在MI小鼠和CoCl_(2)处理的H9c2细胞中miR-31a-5p显著升高,cleaved-caspase 3升高,Bcl-2下降(P均<0.05)。双荧光素酶基因测定结果表明miR-31a-5p mimic与HIF-1α-3'-UTR WT质粒共转染的相对荧光素酶活性降低(P<0.000 1);miR-31a-5p mimic可降低CoCl_(2)诱导的H9c2细胞中HIF-1α的表达,并增加细胞凋亡蛋白水平(P均<0.05),而miR-31a-5p起到相反的作用。 结论 miR-31a-5p 可通过靶向HIF-1α促细胞凋亡加重心肌缺血损伤。Objective To investigate the expression of miR-31a-5p in myocardial infarction(MI)mice and its potential mechanism.Methods A dataset was downloaded from the gene expression database,and miR-31a-5p and its predicted target gene hypoxia-inducible factor-1α(HIF-1α)were screened using bioinformatics methods.The MI model was established by ligating the left anterior descending branch of the coronary artery in C57BL/6J male mice which were randomly divided into sham and MI groups(n=6 in each group).The in vitro hypoxic cell model was induced by treatment of H9c2 cells with cobalt chloride(CoCl_(2))and divided into a control group,a model group,a NC group,a miR-31a-5p mimic group and a miR-31a-5p inhibitor group.The degree of myocardial tissue fibrosis was stained by Masson and analyzed.The expression levels of miR-31a-5p and HIF-1αmRNA in mouse myocardial tissues and H9c2 cells were detected by qRT-PCR.Western blotting was used to detect the expression levels of B-cell lymphoma 2(Bcl-2),cleaved-caspase 3 apoptotic protein in mouse myocardial tissues and HIF-lαand apoptotic protein in H9c2 cells,respectively.The dual luciferase reporter gene assay was used to verify the targeting relationship between miR-31a-5p and HIF-la.Results Masson staining showed significantly increased fibrosis in MI mice(P<0.0001);miR-31a-5p,cleaved-caspase 3 were significantly elevated and Bcl-2 was decreased in MI mice and CoCl,treated H9c2(P<0.05).The results of dual luciferase reporter assay showed that the relative luciferase activity of miR-31a-5p mimic cotransfected with HIF-1α-3'-UTR WT plasmid was reduced(P<0.0001);miR-31a-5p mimic decreased HIF-1αexpression and increased apoptotic protein levels in CoCl_(2) induced H9c2 cells(both P<0.05),while miR-3la-5p exerted the opposite effect.Conclusionn miR-3la-5p can aggravate apoptosis in myocardial ischemia by targeting HIF-1α.

关 键 词：miR-31a-5p 心肌细胞 缺氧诱导因子-1Α 心肌缺氧 细胞凋亡 实验研究 

分 类 号：R542.22[医药卫生—心血管疾病]