Objectives: Hyperhomocysteinaemia (Hcy) is an independent risk factor for cardiovascular and cerebrovascular diseases. MicroRNA (miR)-18a-5p is closely related to cardiovascular diseases. This study aims to investigate the effects of miR-18a-5p on homocysteine (Hcy)-induced myocardial cells injury.
Methods: H9c2 cells were transfected with miR-18a-5p mimic/miR-18a-5p mimic negative control (NC) or combined with Hcy for intervention, and untreated cells were set as a control group. The transfection efficiency was verified by real-time RT-PCR, and cell counting kit-8 (CCK-8) assay was used to determine cell viability. Flow cytometry was used to detect apoptosis and reactive oxygen species (ROS) levels. Western blotting was performed to measure the protein levels of microtubule-associated protein 1 light chain 3 (LC3)-I, LC3-II, Beclin1, p62, Bax, Bcl-2, and Notch2. Dual luciferase reporter assay was used to detect the interaction of miR-18a-5p with Notch2.
Results: Compared with the control, treatment with Hcy or transfection with miR-18a-5p mimic alone, or combined treatment with Hcy and miR-18a-5p mimic/miR-18a-5p mimic NC significantly reduced the H9c2 cell viability, promoted apoptosis and ROS production, up-regulated the expressions of Bax and Beclin, down-regulated the expressions of Bcl-2, p62, and Notch2, and increased the ratio of LC3-II/LC3-I (all P<0.05). Compared with the combined intervention of miR-18a-5p mimic NC and Hcy group, the above indexes were more significantly changed in the combined intervention of miR-18a-5p mimic and Hcy group, and the difference between the 2 groups was statistically significant (all P<0.05). There is a targeted binding between Notch2 and miR-18a-5p.
Conclusions: MiR-18a-5p could induce autophagy and apoptosis via increasing ROS production in cardiomyocytes, and aggravate Hcy-induced myocardial injury. Notch2 is a target of miR-18a-5p.
目的: 高同型半胱氨酸(homocysteine，Hcy)水平是心脑血管疾病的独立危险因素。MiR-18a-5p是微RNA(microRNA，miR)家族中的重要成员，与心血管疾病密切相关。本研究旨在探讨miR-18a-5p对Hcy损伤心肌细胞的影响。方法: 对H9c2细胞进行miR-18a-5p模拟物(mimic)/miR-18a-5p mimic阴性对照(negative control，NC)转染或与Hcy联合干预，另设未处理细胞作为对照组。采用实时反转录聚合酶链反应(real-time reverse transcription PCR，real-time RT-PCR)验证转染效率，细胞计数试剂盒-8(cell counting kit-8，CCK-8)法检测细胞活力，流式细胞术检测细胞凋亡率和细胞中活性氧(reactive oxygen species，ROS)水平，蛋白质印迹法检测微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3，LC3)-I、LC3-II、Beclin1、p62、Bax、Bcl-2和Notch2的蛋白质表达水平，双荧光素酶报告分析检测miR-18a-5p与Notch2的相互作用。结果: 与对照组相比，Hcy或转染miR-18a-5p mimic单独处理，或转染miR-18a-5p mimic/miR-18a-5p mimic NC与Hcy联合干预均显著降低H9c2细胞的活力，增加H9c2细胞的凋亡率和ROS的生成，上调Bax和Beclin1的蛋白质表达水平，下调Bcl-2、p62和Notch2的蛋白质表达水平，同时使LC3-II/LC3-I值增加(均P<0.05)。与miR-18a-5p mimic NC和Hcy联合干预相比，miR-18a-5p mimic和Hcy联合干预的H9c2细胞上述指标的改变更显著，且2组之间差异有统计学意义(均P<0.05)。Notch2与miR-18a-5p存在靶向结合。结论: MiR-18a-5p通过增加心肌细胞内ROS的生成，诱导自噬和凋亡，加重Hcy诱导的心肌损伤。Notch2是miR-18a-5p的作用靶点。.
Keywords: Notch2; autophagy; homocysteine; miR-18a-5p; myocardial injury; reactive oxygen species.