微小核糖核酸-21对过氧化氢诱导C-kit＋心脏干细胞凋亡的作用及机制

Effect and related mechanism of miRNA-21 on hydrogen peroxide-induced C-kit＋ cardiac stem cells apoptosis

目的探讨微小核糖核酸－21（miRNA－21）对过氧化氢（H2O2）诱导的C－kit＋心脏干细胞凋亡的作用及其机制。方法采用酶消化法结合免疫磁珠分选法培养C－kit＋心脏干细胞，然后将C－kit＋心脏干细胞分为对照组（将miRNA－21模拟物阴性对照转染至C－kit＋心脏干细胞48 h）、模拟物组（将miRNA－21模拟物转染至C－kit＋心脏干细胞48 h）、对照＋ H2O2组（将miRNA－21模拟物阴性对照转染至C－kit＋心脏干细胞48 h后，以100 μmol H2O2处理2 h）和模拟物＋ H2O2组 （将miRNA－21模拟物转染至C－kit＋心脏干细胞48 h后，以100 μmol H2O2处理2 h）。采用RT－PCR检测各组miRNA－21的mRNA表达水平；采用流式细胞仪检测各组细胞凋亡情况；采用Western blot检测各组促凋亡相关蛋白（Caspase－3和Bax）和抗凋亡蛋白（Bcl－2）的蛋白表达水平。结果（1）模拟物组的miRNA－21 mRNA表达水平高于对照组（P〈0.05）; 对照＋H2O2组的miRNA－21 mRNA表达水平低于对照组（P〈0.05）；模拟物＋ H2O2 组的miRNA－21表达水平低于模拟物组，而高于对照＋H2O2组（P均〈0.05）。（2）对照组与模拟物组的细胞早期凋亡率差异无统计学意义[（4.57±3.45）%比（5.13±3.21）%，P〉0.05]；对照＋H2O2组细胞早期凋亡率高于对照组[（79.07±5.75）%比（4.57±3.45）%，P〈0.05]; 模拟物＋H2O2组细胞早期凋亡率高于模拟物组[（30.27±1.36）%比（5.13±3.21）%，P〈0.05]，而低于对照＋H2O2组[（30.27±1.36）%比（79.07±5.75）%，P〈0.05]。（3）对照组与模拟物组的Caspase－3、Bax和Bcl－2的蛋白表达水平差异均无统计学意义（P均〉0.05）；对照＋H2O2组Caspase－3和Bax的蛋白表达水平均高于对照组（P均〈0.05），两组之间的Bcl－2蛋白表达水平差异无统计学意义（P〉0.05）；模拟物＋ H2O2 组的Caspase－3和Bax蛋白表达水平均低于对照＋H2O2组（P均〈0.05），而Bcl－2的蛋白表达水平高于对照＋H2O2组（P〈0.05）。结论过表达miRNA－21可通过降低细胞内促凋亡相关蛋白表达和提高抗凋亡蛋白表达，减轻氧化应激所致C－kit＋心脏干细胞凋亡。

ObjectiveTo explored the effect and related mechanism of miRNA-21 on hydrogen peroxide-induced C-kit＋ cardiac stem cells apoptosis. MethodsC-kit＋ cardiac stem cells were isolated from SD rats by the methods of enzyme digestion and magnetic bead. Cells were divided into the following experimental groups： （1） negative control mimics （NCM）group： cells were transfected with negative control miRNA-21 mimics for 48 hours; （2）mimics group： cells were transfected with miRNA-21 mimics for 48 hours; （3） NCM＋ H2O2 group： negative control miRNA-21 mimics were transfected into cells for 48 hours and then treated with 100 μmol H2O2 for 2 hours; （4）mimics＋ H2O2 group： miRNA-21 mimics were transfected into cells for 48 hours and then treated with 100 μmol H2O2 for 2 hours. mRNA of miRNA-21 was detected by RT-PCR. The apoptosis rate of C-kit＋ cardiac stem cells was determined using the annexin V-FITC/PI staining assay. Western blot was employed to measure the expression of apoptosis related proteins（Caspase-3, Bax, and Bcl-2）. Results（1） Compared with the NCM group, the mRNA expression level of miRNA-21 was significantly up-regulated in mimics group, while obviously down-regulated in NCM＋ H2O2 group（all P〈0.05）. Compared with the mimics group, the mRNA expression levels of miRNA-21 in mimics＋ H2O2 group was significantly downregulated （P〈0.05）, but remarkably upregulated compared with the NCM＋ H2O2 group（P〈0.05）. （2） Flow cytometry results indicated that the early apoptosis rates were similar between the NCM group and mimics group （（4.57±3.45）% vs. （5.13±3.21）%, P〉0.05）. Compared with the NCM group, the early apoptosis rates were remarkably increased （（79.07±5.75）% vs.（4.57±3.45）%, P〈0.05） in NCM＋ H2O2 group. Compared with the mimics group, the early apoptosis was significantly up-regulated in the mimics＋ H2O2 group （（30.27±1.36）% vs.（5.13±3.21）%, P〈0.05）, which were further down-regulated in mimics＋ H2O2 group compared with the NCM＋ H2O2 group （（30.27±1.36）% vs.（79.07±5.75）%, P〈0.05）. （3） Western blot results showed similar protein expression of Caspase-3, Bax and Bcl-2 between NCM group and mimics group（all P〉0.05）. Compared with the NCM group, the Caspase-3 and Bax protein expression was significantly increased in NCM＋ H2O2 group （all P〈0.05）, but the protein expression level of Bcl-2 was similar between the 2 groups（P〉0.05）. The Caspase-3 and Bax protein expression was markedly decreased, while Bcl-2 apparently increased in the mimics＋ H2O2 group compared with the NCM＋ H2O2 group（all P〈0.05）. ConclusionOverexpression of miRNA-21 protects the C-kit＋ cardiac stem cells from apoptosis caused by oxidative stress through downregulating proapoptotic and upregulating the antiapoptotic proteins.