摘要
目的探讨miR-494在阿霉素(DOX)诱导大鼠心肌细胞损伤中的作用及机制。方法将大鼠H9C2细胞分为4组:对照组、DOX组、DOX+阴性对照(NC)模拟物组、DOX+miR-494模拟物组;除对照组,其余3组均采用5μmol/L DOX与细胞共孵育法构建心肌细胞损伤模型;后两组分别进行NC模拟物、miR-494模拟物转染。采用细胞计数试剂盒8法检测细胞活力,实时荧光定量聚合酶链反应检测miR-494、磷酸酶和张力蛋白同系物(PTEN)m RNA表达水平,原位末端转移酶标记染色法检测细胞凋亡率,蛋白质印迹法检测B淋巴细胞瘤-2(Bcl-2)、Bcl-2关联X蛋白(Bax)、PTEN、磷酸化磷脂酰肌醇3-激酶(p-PI3K)、磷酸化蛋白激酶B(p-Akt)蛋白表达水平,酶联免疫吸附试验检测乳酸脱氢酶(LDH)、肌酸激酶同工酶(CK-MB)含量;采用双荧光素酶报告基因实验验证miR-494与PTEN的相互作用。结果与对照组比较,DOX组H9C2细胞凋亡率、Bax蛋白表达水平以及LDH、CK-MB含量均明显升高(均P<0.01),而细胞活力、Bcl-2蛋白表达水平均明显降低(均P<0.01);与DOX+NC模拟物组比较,DOX+miR-494模拟物组H9C2细胞凋亡率、Bax蛋白表达水平以及LDH、CK-MB含量均明显降低(均P<0.01),而细胞活力、Bcl-2蛋白表达水平均明显升高(均P<0.01)。PTEN与miR-494存在结合位点;PTEN-WT的荧光素酶活性被miR-494模拟物明显抑制(P=0.010),而PTEN-MUT的荧光素酶活性不受miR-494模拟物影响(P=0.707)。与对照组比较,DOX组H9C2细胞中p-PI3K、p-Akt蛋白表达水平均明显降低(均P<0.01);与DOX+NC模拟物组比较,DOX+miR-494模拟物组H9C2细胞中p-PI3K、p-Akt蛋白表达水平均明显升高(均P<0.01)。结论miR-494可能通过靶向抑制PTEN表达来调控PI3K/Akt信号通路,以改善DOX诱导的心肌细胞损伤。
Objective To investigate the role and mechanism of mi R-494 in doxorubicin(DOX)induced cardiomyocyteinjury in rats.Methods H9C2 cells were divided into four groups:control group,DOX group,DOX+negative control(NC)mimics group,and DOX+mi R-494 mimics group.Except for the control group,the remaining three groups wereexposed to 5μmol/L DOX to establish a cardiomyocyte injury model,the latter two groups were transfected with NC mimics and mi R-494 mimics,respectively.Cell viability was detected using the cell counting kit-8.Quantitative real-time polymerase chain reaction was used to detect the m RNA expression levels of mi R-494,phosphatase and tensin homolog(PTEN).Terminal-deoxynucleotidyl transferase-mediated d UTP-biotin nick end labeling was used to detect cell apoptosis rate.Western blot was used to detect the expression levels of B-cell lymphoma-2(Bcl-2),Bcl-2associated X protein(Bax),PTEN,phosphorylated PI3K(p-PI3K),and phosphorylated Akt(p-Akt).Enzyme-linked immunosorbent assay was used to detect the levels of lactate dehydrogenase(LDH)and creatine kinase-MB(CK-MB).Verify the interaction between mi R-494 and PTEN using dual-luciferase reporter assay.Results Compared with the control group,the apoptosis rate,Bax protein expression level,LDH and CK-MB content of H9C2 cells in the DOX group were significantly increased(all P<0.01),while cell viability and Bcl-2 protein expression level were significantly reduced(all P<0.01).Compared with the DOX+NC mimetic group,the apoptosis rate,Bax protein expression level,LDH and CK-MB content of H9C2 cells in the DOX+mi R-494 mimetic group were significantly reduced(all P<0.01),while cell viability and Bcl-2 protein expression level were significantly increased(all P<0.01).PTEN has binding sites with mi R-494.The luciferase activity of PTEN-WT was significantly reduced by the mi R-494 mimetic(P=0.010),while the luciferase activity of PTEN-MUT was not affected by the mi R-494 mimetic(P=0.707).Compared with the control group,the expression levels of p-PI3K and p-Akt proteins in H9C2 cells in the DOX group were significantly reduced(all P<0.01).Compared with the DOX+NC mimetic group,the expression levels of p-PI3K and p-Akt proteins in H9C2 cells in the DOX+mi R-494 mimetic group were significantly increased(all P<0.01).Conclusion mi R-494 may regulate the PI3K/Akt signaling pathway by targeting and inhibiting PTEN expression,thus ameliorating DOX induced cardiomyocyte injury.
作者
沈盛晖
叶建华
吴相忠
冯频频
彭鹏
SHEN Shenghui;YE Jianhua;WU Xiangzhong;FENG Pinpin;PENG Peng(Department of Cardiology,Tongde Hospital of Zhejiang Province(Zhejiang Academy of Traditional Chinese Medicine),Hangzhou 310012,China;不详)
出处
《心电与循环》
2024年第4期316-322,I0001,共8页
Journal of Electrocardiology and Circulation
基金
浙江省基础公益研究计划项目(LGF22H020018)
浙江省医药卫生科技计划项目(2022KY701)。
