MicroRNA-34a通过下调Notch1的表达促进心肌细胞凋亡

目的：探讨microRNA-34a（miR-34a）对心肌细胞凋亡的影响及其机制。方法：体外培养大鼠心肌细胞H9C2，分别转染miR-34a mimics、miR-34a inhibitor、随机合成的miR-NC片段、Notch1的siRNA、随机合成的siRNA-NC片段，将实验分为6 组：normal组、miR-34a组、miR-inhibitor组、miR-NC组、miRinhibitor+siRNA-Notch1组和miR-inhibitor+siRNA-NC组。RT-qPCR检测各组miR-34a的表达量，Westernbolt技术检测各组中caspase-3 和Notch1的表达量，流式细胞技术检测各组细胞的凋亡率。双荧光素酶报告实验鉴定Notch1为miR-34a的靶基因。结果：双荧光素酶报告实验证实miR-34a和Notch1间存在靶向关系。与miR-NC组比较，miR-34a组的miR-34a和caspase-3的表达量明显增多，Notch1的表达量明显减少，细胞凋亡率明显升高（P ＜0.01）。与miR-NC组比较，miR-inhibitor组的miR-34a和caspase-3的表达量明显减少，Notch1的表达量明显增多，细胞凋亡率明显减低（P ＜0.01）。与miR-inhibitor+siRNA-NC组比较，miRinhibitor+siRNA-Notch1组的Notch1的表达量明显降少，caspase-3的表达量明显减少，细胞凋亡率明显减低（P ＜0.01）。结论：miR-34a促进心肌细胞凋亡，其作用机制与靶向负调控Notch1有关。

Cardiomyocyte apoptosis induced by microRNA-34a through down-regulating Notch1

Objective: To investigate the effect of microRNA-34a (miR-34a) on myocardial apoptosis and its underlying mechanism. Methods: Rat cardiomyocyte line (H9C2) was transfected with miR-34a mimics,miR-34a inhibitor, randomly synthesized miR-NC fragments, siRNA of Notch1, and randomly synthesized siRNA-NC fragments, respectively. The experiment was divided into six groups: normal group, miR-34a group,miR-inhibitor group, miR-NC group, miR-inhibitor+sirNA-Notch1 group, and miR-inhibitor+sirNA-NC group.Real-time quantitative PCR was used to analyze the expression of miR-34a. Western bolt was used to detect the expression of Caspase-3 and Notch1. The apoptosis rate of cardiomyocyte in each group was measured by Flow cytometry. Notch1 was identified as a target gene of miR-34a by dual luciferase assay. Results: Dual luciferase assay confirmed the targeting relationship between miR-34a and Notch1. Compared with the miR-NC group, the expression of miR-34a and caspase-3 was significantly increased, while the expression of Notch1 was obviously decreased, and the apoptosis rate of cardiomyocytes was apparently increased in the miR-34a group (P<0.01).Compared with the miR-NC group, the expression of miR-34a and Caspase-3 was significantly decreased, while the expression of Notch1 was obviously increased, and the apoptosis rate of cardiomyocytes was apparently decreased in the miR-inhibitor group (P<0.01). Compared with miR-inhibitor+siRNA-NC group, the expression of Notch1 and caspase-3 was significantly decreased, and the apoptosis rate of cardiomyocytes was apparently decreased in the miR-inhibitor+siRNA-Notch1 group (P<0.01). Conclusion: MiR-34a induces cardiomyocyte apoptosis through down-regulating Notch1.