microRNA-206对低氧诱导心肌细胞凋亡的作用及其可能机制  

Effect of microRNA-206 on hypoxia-induced cardiomyocyte apoptosis and its possible mechanism

作　　者：陈昆 向阳 吴冰 吴三五 CHEN Kun;XIANG Yang;WU Bing(Department of Cardiology,Renmin hospitalHubei University of Medicine,Shiyan Hubei 442000,China)

机构地区：[1]十堰市人民医院(湖北医药学院附属人民医院)心血管内科,湖北十堰442000

出　　处：《临床和实验医学杂志》2020年第11期1175-1179,共5页Journal of Clinical and Experimental Medicine

基　　金：十堰市医药卫生科技研究项目(编号:2016SY107)。

摘　　要：目的探讨microRNA-206(miR-206)在低氧诱导的原代心肌细胞凋亡中的作用及可能的作用机制。方法将心肌细胞分成六组,正常组、低氧组、miR-206模拟物组、miR-206模拟物-NC组、miR-206抑制剂组和miR-206抑制剂-NC组,其中正常组心肌细胞未转染,正常培养,低氧组心肌细胞未转染,经过低氧处理,miR-206模拟物组、miR-206模拟物-NC组心肌细胞经低氧处理后分别转染miR-206模拟物及其阴性对照序列,miR-206抑制剂组和miR-206抑制剂-NC组心肌细胞在低氧处理后分别转染miR-206抑制剂及其阴性对照序列。设置不同低氧处理时间(0 h、12 h、24 h和48 h)处理心肌细胞,同时正常组细胞正常培养,四甲基偶氮唑盐微量酶反应比色法(MTT)检测细胞增殖情况,qRT-PCR检测miR-206表达情况,采用流式细胞术、免疫组化检测和Western blot法检测细胞凋亡情况。MTT检测六组心肌细胞增殖变化情况,流式细胞术检测细胞凋亡改变情况,qRT-PCR检测表皮生长因子受体(EGFR)-低氧诱导因子1α(HIF-1α)信号通路mRNA表达情况,Western Blot检测EGFR-HIF-1α信号通路蛋白表达情况。结果MTT结果显示心肌细胞随低氧处理时间增加增殖呈下降趋势,低氧处理24 h和48 h细胞增殖显著低于正常组(P<0.05);免疫组化检测结果可知,低氧诱导48 h后,心肌细胞内凋亡蛋白cleaved-caspas3显著增加;流式细胞仪检测结果可知低氧诱导24 h、48 h后细胞凋亡呈时间依赖性增加;Western blot鉴定并证实细胞凋亡相关蛋白表达增加及抗凋亡蛋白表达的下降;心肌细胞随低氧处理时间增加miR-206表达呈上升趋势,处理24 h和48 h的miR-206表达量显著高于正常组(P<0.05)。miR-206模拟物转染组细胞增殖显著增加,凋亡明显下降,miR-206抑制剂组增殖显著下降,凋亡显著增加(P<0.05)。qRT-PCR法检测发现干扰后各组EGFR、HIF-1α和胰岛素样生长因子1(IGF-1)的mRNA水平无变化,但Western blot结果显示miR-206模�Objective To investigate the expression level of miR-206 on cardiomyocyte cells and effect on cell apoptosis and its possible mechanism.Methods The myocardial cells were divided into six groups,including the normal group,the hypoxia group,the miR-206 mimics group and the miR-206 mimics-NC group,the miR-206 inhibitor group and miR-206 inhibitor-NC group.The miR-206 mimics group and miR-206 mimics-NC group cells were transfected with miR-206 mimics and negative control sequences,the miR-206 inhibitor group and miR-206 inhibitor-NC group cells were transfected with miR-206 inhibitor and negative control sequences after hypoxia induction,the hypoxia group was induced by hypoxia,the normal group was cultured with the normal culture of the non-transfected cardiomyocytes.The myocardial cells were treated at different hypoxia-induced time(0 h,12 h,24 h and 48 h),while the myocardial cells in normal group were cultured as usual,MTT was used to detect the proliferation of cardiomyocytes,qRT-PCR was used to detect the expression of miR-206,and flow cytometry,immunohistochemical method and Western blot were used to determine the apoptosis of cardiomyocytes.MTT was used to detect myocardial cell proliferation status of the above six groups,and flow cytometry was used to detect the apoptosis of cardiomyocytes.qRT-PCR was used to detect the mRNA expression of EGFR-HIF-1αsignaling pathway,and Western blot was used to detect the expression of EGFR-HIF-1αsignaling pathway protein.Results MTT results showed that cell proliferation was downward trend as the hypoxia-induced time increased,cell proliferation after hypoxia induction for 24h and 48h were significantly lower than that of the normal control group(P<0.05),and the expression of the miR-206 was just the opposite(P<0.05).The results of immunohistochemical method showed that cleaved-caspas3 expression in myocardial cells after hypoxia induction for 48h was higher than that of the normal group.The results of flow cytometry showed that apoptosis increased in a time-dependent m

关 键 词：心肌细胞 低氧 凋亡 microRNA-206 EGFR-IGF-1-HIF-1α信号通路 

分 类 号：R541.4[医药卫生—心血管疾病]