下调miR-199a-5p对阿霉素诱导的心肌细胞凋亡的影响和机制研究

目的:探讨下调miR-199a-5p对阿霉素诱导的心肌细胞凋亡的影响和机制。方法:将心肌细胞H9C2分成Control组(常规培养的对照细胞)、DOX组(经含1μM阿霉素的细胞培养液培养)、DOX+Anti-miR-NC组(转染inhibitor control,经含1μM阿霉素的细胞培养液培养)和DOX+Anti-miR-199a-5p组(转染miR-199a-5p inhibitor,经含1μM阿霉素的细胞培养液培养)、DOX+Anti-miR-199a-5p+DKK1组(转染miR-199a-5p inhibitor,经含1μM阿霉素和20ng/ml的Wnt/β-catenin信号抑制剂DKK1的细胞培养液培养)。各组细胞处理24h以后,Realtime PCR测定miR-199a-5p表达,CCK-8测定细胞增殖,流式细胞术测定细胞凋亡,Western blot测定C-Caspase-3、β-catenin、c-Myc蛋白表达。结果:与Control组比较,DOX组细胞中miR-199a-5p水平升高,细胞增殖活性下降,细胞凋亡和C-Caspase-3蛋白表达水平升高,β-catenin、c-Myc蛋白表达水平降低(P均<0.01)。与DOX+Anti-miR-NC组比较,DOX+Anti-miR-199a-5p组细胞中miR-199a-5p水平降低,细胞增殖活性升高,细胞凋亡水平和C-Caspase-3蛋白表达水平降低,β-catenin、c-Myc蛋白表达水平升高(P均<0.01)。与DOX+Anti-miR-199a-5p组相比,DOX+Anti-miR-199a-5p+DKK1组心肌细胞存活率降低,细胞凋亡率升高,细胞中C-Caspase-3蛋白表达水平升高,β-catenin、c-Myc蛋白表达水平降低(P均<0.01)。结论:下调miR-199a-5p通过激活Wnt/β-catenin信号抑制阿霉素诱导的心肌细胞凋亡。

The Effect and Mechanism of Down-regulation of miR-199a-5p on Doxorubicin-induced Cardiomyocyte Apoptosis

Objective:To investigate the effect and mechanism of down-regulation of miR-199a-5p on doxorubicin-induced cardiomyocyte apoptosis.Method:Cardiomyocytes H9C2 were divided into control group(conventional cultured control cells),DOX group(cultured in cell culture medium containing 1μM doxorubicin),DOX+Anti-miR-NC group(transfected with inhibitor control,cultured in cell culture medium containing 1μM doxorubicin),DOX+Anti-miR-199a-5p group(transfected with miR-199a-5p inhibitor,cultured in cell culture medium containing 1μM doxorubicin),DOX+Anti-miR-199a-5p+DKK1 group(transfected with miR-199a-5p inhibitor,cultured in cell culture medium containing 1μM doxorubicin and 20ng/ml Wnt/β-catenin signal inhibitor DKK1).After 24h,Realtime PCR method was used to measure the expression of miR-199a-5p,CCK-8 was used to measure cell proliferation,flow cytometry was used to measure cell apoptosis,and Western blot was used to measure the expression of C-Caspase-3,β-catenin and c-Myc protein.Results:Compared with the Control group,the level of miR-199a-5p in the DOX group was increased,cell proliferation activity was decreased,cell apoptosis and C-Caspase-3 protein expression were increased,β-catenin and c-Myc protein expression were reduced.Compared with DOX+Anti-miR-NC group,miR-199a-5p level was decreased,cell proliferation activity was increased,cell apoptosis level and C-Caspase-3 protein expression were decreased,β-catenin,c-Myc protein expression were increased in DOX+Anti-miR-199a-5p group.Compared with the DOX+Anti-miR-199a-5p group,the survival rate of cardiomyocytes was decreased,the apoptosis rate was increased,and the expression of C-Caspase-3 protein was increased,the expression ofβ-catenin and c-Myc protein were decreased in the DOX+Anti-miR-199a-5p+DKK1 group.Conclusion:Down-regulation of miR-199a-5p inhibits doxorubicin-induced cardiomyocyte apoptosis by activating Wnt/β-catenin signal.