miR-499通过抑制calcineurin抑制缺血/再灌注心肌细胞的凋亡  

MiR-499 inhibits apoptosis of ischemia/reperfusion cardiomyocytes by inhibiting calcineurin

作　　者：吴静[1] 聂祖琼 尹琬凌 WU Jing;NIE Zuqiong;YIN Wanling(Department of Geriatrics,Central Hospital of Wuhan,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430014,China)

机构地区：[1]华中科技大学同济医学院附属武汉市中心医院老年病科,武汉430014

出　　处：《山西医科大学学报》2023年第7期932-938,共7页Journal of Shanxi Medical University

基　　金：武汉市卫生计生委科研计划资助项目(WX21C17)。

摘　　要：目的 探讨miR-499对钙调磷酸酶(calcineurin,CaN)的调控作用以及在缺血/再灌注(ischemia/repefusion,I/R)损伤心肌细胞中的作用机制。方法 将大鼠心肌细胞H9c2分成6组:对照组、I/R组、I/R+mimics组(转染miR-499 mimics后构建I/R细胞模型)、I/R+NC组(转染miR-NC后构建I/R细胞模型)、I/R+mimics+CsA组(转染miR-499 mimics后加入CaN抑制剂CsA处理,最后构建I/R细胞模型)和I/R+NC+CsA组(转染miR-NC后加入CsA处理,最后构建I/R细胞模型)。CCK-8检测各组细胞增殖能力,Hochest33258染色观察各组细胞形态的变化,TUNEL检测各组细胞凋亡情况,qRT-PCR检测各组细胞中miR-499、CaN和凋亡相关(Bcl-2、Bax、Caspase-3)基因的表达,Western blot检测各组细胞中CaN和凋亡相关(Bcl-2、Bax、Caspase-3)蛋白的表达。结果 与对照组相比,I/R组细胞增殖能力显著降低(P<0.01),细胞致密浓染,呈亮蓝色,凋亡细胞增多,细胞中miR-499、Bcl-2 mRNA和蛋白表达水平显著降低(P<0.01),CaN、Bax和Caspase-3 mRNA和蛋白水平显著升高(P<0.01);与I/R+NC组相比,I/R+mimics组和I/R+NC+CsA组细胞增殖能力均显著升高(P<0.01),致密浓染的细胞核均有所减少,凋亡细胞减少,细胞中miR-499、Bcl-2 mRNA和蛋白表达水平显著升高(P<0.05),CaN、Bax和Caspase-3 mRNA和蛋白水平显著降低(P<0.05);与I/R+NC+CsA组相比,I/R+mimics+CsA组上述作用更加显著。结论 miR-499能够抑制CaN的表达,抑制I/R损伤心肌细胞的凋亡,减轻心肌细胞损伤。Objective To investigate the regulatory effect of miR-499 on calcineurin(CaN)and the role of miR-499 in ischemia/reperfusion(I/R)injury myocardial cells.Methods Rat cardiomyocytes H9c2 were divided into six groups:control group,I/R group,I/R+mimics group(transfection with miR-499 mimics and then I/R),I/R+NC group(transfection with miRNC and then I/R),I/R+mimics+CsA group(I/R after transfection with miR-499 mimics and CsA treatment)and I/R+NC+CsA group(I/R after transfection with miR-NC and CsA treatment).The cell proliferation was detected by CCK-8,the cell morphological changes were observed by Hochest33258 staining,the cell apoptosis was detected by TUNEL,the expression levels of miR-499,CaN and apoptosis-related genes(Bcl-2,Bax,Caspase-3)in cells were detected by qRT-PCR,and the expression levels of CaN and apoptosis-related proteins(Bcl-2,Bax,Caspase-3)in the cells were detected by Western blot.Results Compared with control group,the cell proli-feration ability was significantly decreased in I/R group(P<0.01),the cells were densely stained with bright blue,the apoptotic cells were increased,and the miR-499 and Bcl-2 mRNA and protein expression levels in the cells were significantly decreased(P<0.01),and the mRNA and protein levels of CaN,Bax and Caspase-3 were significantly increased(P<0.01).Compared with I/R+NC group,the cell proliferation ability was significantly increased in I/R+mimics group and I/R+NC+CsA group(P<0.01),the apoptotic cells were decreased,the miR-499 and Bcl-2 mRNA and protein expression levels were significantly increased(P<0.05),the mRNA and protein levels of CaN,Bax and Caspase-3 were significantly decreased(P<0.05).The above effects were more significant in I/R+mimics+CsA group than in I/R+NC+CsA group.Conclusion miR-499 can inhibit the expression of CaN,inhibit the apoptosis of I/R injury cardiomyocytes and reduce the damage of cardiomyocytes.

关 键 词：miR-499 钙调磷酸酶 缺血/再灌注 凋亡 心肌细胞 

分 类 号：R542.2[医药卫生—心血管疾病]