摘要
目的:探讨miR-19b对阿霉素(DOX)诱导的H9c2心肌细胞损伤的影响。方法:采用DOX(250 ng/ml)诱导大鼠H9c2心肌细胞,构建心肌细胞损伤模型。通过MTT检测DOX作用12、24、48 h后的细胞活力;miR-19b mimic和miR-19b inhibitor转染DOX诱导后的大鼠H9c2细胞,采用RT-PCR检测转染后细胞中miR-19b的表达情况,流式细胞术检测细胞内ROS水平和线粒体膜电位水平,ATP检测试剂盒检测细胞内ATP水平,Western blot检测线粒体凋亡相关蛋白Bad、Bcl-2、Caspase-9、Caspase-3、细胞色素C(Cyt-C)和p53的表达。结果:MTT结果显示DOX处理后,H9c2细胞的存活率显著降低(P<0.05);miR-19b mimic/inhibitor转染可显著上调或下调H9c2细胞中miR-19b的表达(P<0.05)。与DOX组相比,miR-19b mimic组ROS水平降低,ATP水平升高(P<0.05)同时细胞凋亡减少,具体表现为线粒体低电位细胞比例减少,促凋亡相关蛋白Bad、Caspase-9、Caspase-3、Cyt-C和p53表达量降低(P<0.05),抑制凋亡蛋白Bcl-2的表达量升高(P<0.05)。而miR-19b inhibitor组ROS水平升高,ATP水平降低(P<0.05)同时细胞凋亡增加,具体表现为线粒体低电位细胞比例增加,Bad、Caspase-9、Caspase-3、Cyt-C和p53蛋白表达升高(P<0.05),Bcl-2蛋白表达降低(P<0.05)。结论:miR-19b过表达能有效缓解DOX诱导的H9c2心肌细胞线粒体凋亡。
Objective:To investigate the effect of miR-19b on damage of myocardial cell line H9c2 induced by doxorubicin(DOX).Methods:The H9c2 cells were administrated with DOX(250 ng/ml)to constructed DOX-induced myocardial cell injury model.The cell viability of H9c2 cells after treated with DOX for 12,24,and 48 h was measured using MTT.The DOX administrated H9c2 cells were then transfected with miR-19b mimic/inhibitor.After transfection,RT-PCR was performed to detect the expression of miR-19b;flow cytometry was performed to evaluate the level of ROS and mitochondrial membrane potential;Western blot was performed to measure the proteins expression of Bad,Bcl-2,Caspase-9,Caspase-3,Cytochrome C(Cyt-C),and p53.In addition,the ATP level was detected using the corresponding kit.Results:The results of MTT demonstrated that DOX significantly increased viability of H9c2 cells(P<0.05);miR-19b mimic/inhibitor transfection obviously up-regulated/down-regulated the expression of miR-19b(P<0.05).Compared to DOX group,the ROS level in miR-19b mimic group was decreased,the ATP level was increased(P<0.05).In addition,the cell apoptosis was decreased,as demonstrated by the decrease in the proportion of cells with low mitochondrial membrane potential,as well as the expression of pro-apoptotic proteins Bad,Caspase-9,Caspase-3,Cyt-C,and p53(P<0.05),and the decrease of the expression of anti-apoptoic protein Bcl-2(P<0.05).Whereas,these results in miR-19b inhibitor group showed the reverse trend.Conclusion:The overexpression of miR-19b can effectively alleviate DOX-induced mitochondrial apoptosis of H9c2 cardiomyocyte.
作者
周琦
刘敏
骆春艳
明强(指导)
ZHOU Qi;LIU Min;LUO Chun-Yan;MING Qiang(Department of Cardiovascular Medicine,Tongji Tianyou Hospital,Wuhan 461000,China)
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2020年第17期2077-2081,共5页
Chinese Journal of Immunology
