miRNA-499a-5p靶向CD38对过氧化氢诱导的心肌细胞损伤的影响及其机制研究

Effects of miRNA-499a-5p targeting CD38 on hydrogen peroxide-induced cardiomyocyte injury and the possible mechanism

目的探讨miRNA-499a-5p靶向CD38对过氧化氢(H2O2)诱导的心肌细胞损伤的影响及作用机制。方法体外培养H9c2心肌细胞,采用H2O2诱导心肌细胞损伤模型。实验分为对照组、H2O2组、H2O2+阴性对照(NC)组和H2O2+miRNA-499a-5p组。实时荧光定量PCR(RT-qPCR)和蛋白质免疫印迹(Western blot)法分别检测心肌细胞中miRNA-499a-5p和CD38的表达,噻唑蓝(MTT)法检测心肌细胞存活率,试剂盒检测活性氧簇(ROS)水平和乳酸脱氢酶(LDH)活性,Annexin V-FITC/PI双染法检测心肌细胞凋亡率,Western blot检测B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、磷酸肌醇3-激酶(PI3K)、磷酸化PI3K(p-PI3K)、蛋白激酶B(Akt)和磷酸化Akt(p-Akt)的表达量。双荧光素酶报告基因试验检测miRNA-499a-5p和CD38的靶向关系。结果H2O2组心肌细胞中miRNA-499a-5p、Bcl-2、p-PI3K、p-Akt的表达水平及心肌细胞存活率与对照组相比均明显降低(P<0.05),而CD38和Bax的表达水平、ROS水平、LDH活性及细胞凋亡率均明显升高(P<0.05)。H2O2+miRNA-499a-5p组心肌细胞中miRNA-499a-5p、Bcl-2、p-PI3K、p-Akt的表达水平及心肌细胞存活率与H2O2组相比均明显升高(P<0.05),而CD38和Bax的表达水平、ROS水平、LDH活性及细胞凋亡率均明显降低(P<0.05)。双荧光素酶报告基因试验证实了CD38是miRNA-499a-5p的靶基因。结论miRNA-499a-5p能够通过抑制CD38的表达减轻H2O2诱导的心肌细胞损伤,该作用机制可能与PI3K/Akt信号通路有关。

Objective To investigate the effects of miRNA-499a-5p targeting CD38 on cardiomyocyte injury induced by hydrogen peroxide(H2O2)and the possible mechanism.Methods H9c2 cardiomyocytes were cultured in vitro and induced with H2O2 to establish the cardiomyocyte injury model.Four groups including control,H2O2,H2O2+negative control(NC)and H2O2+miRNA-499a-5p groups were set up.Real-time quantitative PCR(RT-qPCR)and Western blot were used to detect the expression of miRNA-499a-5p and CD38 in cardiomyocytes,respectively.MTT assay was used to measure the survival rates of cardiomyocytes.The levels of reactive oxygen species(ROS)and the activity of lactate dehydrogenase(LDH)were analyzed with test kits.Annexin V-FITC/PI double staining method was used to detect the apoptosis rates of cardiomyocytes.Western blot was used to detect the expression of B-cell lymphoma/leukemia-2(Bcl-2),Bcl-2-related X protein(Bax),phosphoinositide 3-kinase(PI3K),phosphorylated PI3K(p-PI3K),protein kinase B(Akt)and phosphorylated Akt(p-Akt).Dual luciferase reporter assay was used to detect the targeting relationship between miRNA-499a-5p and CD38.Results Compared with the control group,the expression of miRNA-499a-5p,Bcl-2,p-PI3K and p-Akt and the survival rate of cardiomyocytes were significantly reduced in the H2O2 group(P<0.05),while the expression of CD38 and Bax,ROS level,LDH activity and the apoptosis rate were significantly increased(P<0.05).Compared with the H2O2 group,the expression of miRNA-499a-5p,Bcl-2,p-PI3K and p-Akt and the survival rate of cardiomyocytes were significantly increased in the H2O2+miRNA-499a-5p group(P<0.05),while the expression of CD38 and Bax,ROS level,LDH activity and the apoptosis rate were significantly decreased(P<0.05).Dual luciferase reporter assay confirmed that CD38 was a target gene of miRNA-499a-5p.Conclusions miRNA-499a-5p could alleviate H2O2-induced cardiomyocyte injury by inhibiting the expression of CD38 and the PI3K/Akt signaling pathway might be involved.