李倩晓 于勤 那荣妹 刘百亭

[摘要] 目的 研究miR-1與miR-499在心肌细胞增殖与凋亡中的调控作用及其机制。 方法 通过脂质体2000转染试剂将miR-1 mimics、miR-499 inhibitor转染至H9C2心肌细胞。设置空白对照组、H2O2组（未进行转染的H9C2心肌细胞）、干预组A（miR-1 mimics转染的H9C2心肌细胞）、干预组B（miR-499 inhibitor转染的H9C2心肌细胞）、干预组C（miR-1 mimics+miR-499 inhibitor转染的H9C2心肌细胞）。通过H2O2诱导建立H9C2心肌细胞氧化应激模型。采用CCK-8法检测细胞增殖情况，流式细胞仪检测细胞凋亡情况，Western Blot检测Bim、Mcl-1蛋白表达水平。 结果 与空白对照组比较，H2O2组细胞增殖显著降低而细胞凋亡率显著升高，差异有统计学意义（P<0.05）。与H2O2组相比，干预组A、干预组B细胞增殖进一步降低而细胞凋亡率进一步升高，这种改变在干预组C中更加显著，差异有统计学意义（P<0.05）。与空白对照组比较，H2O2组Bim蛋白表达水平明显升高，Mcl-1蛋白表达水平明显降低，差异有统计学意义（P<0.05）。与H2O2组相比，干预组A、干预组B的Bim蛋白表达水平进一步升高，Mcl-1蛋白表达水平进一步降低，这种改变在干预组C中更加显著，差异有统计学意义（P<0.05）。 结论 miR-1与miR-499在心肌细胞增殖与凋亡中发挥重要调控作用，miR-1可能通过上调Bim、下调Mcl-1蛋白表达促进心肌细胞凋亡，miR-499则通过下调Bim、上调Mcl-1蛋白表达抑制心肌细胞凋亡。

[关键词] miR-1;miR-499;调控;心肌细胞;凋亡

[中图分类号] R587.2          [文献标识码] A          [文章编号] 1673-9701（2020）17-0037-04

A study of the regulatory effect of miR-1 and miR-499 on the proliferation and apoptosis of cardiomyocytes and the mechanism

LI Qianxiao1   YU Qin2   NA Rongmei2   LIU Baiting2

1.Department of Cardiology，Zhejiang Integrated Traditional and Western Medicine Hospital，Hangzhou   310000，China; 2.Department of Cardiology，Affiliated Zhongshan Hospital of Dalian University，Dalian   116001，China

[Abstract] Objective To study the regulatory effect of miR-1 and miR-499 on the proliferation and apoptosis of cardiomyocytes and the mechanism. Methods MiR-1 mimics and miR-499 inhibitor were transfected into H9C2 cardiomyocytes by liposome 2000 transfection reagent. Those groups were set up such as the blank control group， the H2O2 group（H9C2 cardiomyocytes not transfected），the intervention group A（H9C2 cardiomyocytes transfected by miR-1 mimics）， the intervention group B（H9C2 cardiomyocytes transfected by miR-499 inhibitor）， and the intervention group C（H9C2 cardiomyocytes transfected by miR-1 mimic+miR-499 inhibitor）. The oxidative stress model of H9C2 cardiomyocytes was established by H2O2 induction. The cell proliferation was detected by the CCK-8 method， the cell apoptosis was detected by flow cytometer，and Bim and Mcl-1 protein expression levels were detected by Western Blot. Results Compared with that in the blank control group， the proliferation of cells in the H2O2 group was significantly reduced while the apoptosis rate of cells was significantly increased， with statistically significant differences（P<0.05）. Compared with that in the H2O2 group，the proliferation of cells in the intervention group A and the intervention group B further decreased while the apoptosis rate of cells in the two groups further increased， and the correspondent changes were more significant in the intervention group C， with statistically significant differences（P<0.05）. Compared with those in the blank control group，the Bim protein expression level in the H2O2 group significantly increased and the Mcl-1 protein expression level significantly decreased， with statistically significant differences（P<0.05）. Compared with those in the H2O2 group， the Bim protein expression level in the intervention group A and the intervention group B further increased and the Mcl-1 protein expression level further decreased， and the correspondent changes were more significant in the intervention group C，with statistically significant differences（P<0.05）. Conclusion MiR-1 and miR-499 play important regulatory roles in the proliferation and apoptosis of cardiomyocytes. MiR-1 may promote the apoptosis of cardiomyocytes by up-regulating Bim protein expression and down-regulating Mcl-1 protein expression， while miR-499 may inhibit the apoptosis of cardiomyocytes by down-regulating Bim protein expression and up-regulating Mcl-1 protein expression.

[Key words] miR-1;miR-499;Regulation;Cardiomyocyte;Apoptosis

心肌细胞凋亡与心脏疾病密切相关，包括心肌缺血再灌注损伤、缺血性心脏病、心力衰竭、心肌病等[1]。近年来，关于微小RNA（microRNA，miRNA）调控心肌细胞凋亡的研究已成为热点，为心脏疾病的诊治提供了重要方向[2]。本研究将miR-1 mimics、miR-499 inhibitor转染至H2O2处理的心肌细胞，探讨miR-1与miR-499在心肌细胞增殖与凋亡过程中的调控作用及其机制，现报道如下。

1 材料与方法

1.1 材料来源

大鼠H9C2心肌细胞株，购自中科院上海细胞库;胎牛血清、DMEM培养基、0.05%胰酶/EDTA（美国Hycloneo公司）;miR-1 mimics、miR-499 inhibitor、竞争性短核糖核苷酸阴性对照序列（scramble-NC）、miR-499、miR-1和U6引物序列（上海吉凯基因化学技术有限公司）;H2O2、CCK-8、Annexin V-FITC/PI细胞凋亡检测试剂盒（中国碧云天试剂公司）;β-actin、Bim、Mcl-1单抗（美国Epitmics公司）;ECL化学发光试剂盒（美国millipore公司）;脂质体2000转染试剂（美国Invitrogen公司）;二喹啉甲酸（BCA）试剂盒（美国Thermo公司）;miRNeasy Serum/plasma Kit、miScript SYBR Green PCR Kit、RNase-free ddH2O、逆转录试剂盒（德国QIAGEN公司）。RT-PCR检测仪（美国ABI公司），流式细胞仪（美国BD公司），酶标仪（美国Bio-Tek公司）。……