MiR-18a-5p通过介导自噬加重同型半胱氨酸诱导的心肌损伤  

MiR-18a-5p aggravates homocysteine-induced myocardial injury via autophagy

作　　者：尹绢[1] 胡龙龙 韩雪玲 陈露 余玲玲[3] 卢寅辉[1] YIN Juan;HU Longlong;HAN Xueling;CHEN Lu;YU Lingling;LU Yinhui(Department of Geriatrics,Jiangxi Provincial People’s Hospital(First Affliated Hospital of Nanchang Medical College),Nanchang 330006;Department of Cardiology,Second Affiliated Hospital of Nanchang University,Nanchang 330008;Department of Rehabilitation,Second Affiliated Hospital of Nanchang University,Nanchang 330008,China)

机构地区：[1]江西省人民医院(南昌医学院第一附属医院)老年科,南昌330006 [2]南昌大学第二附属医院心内科,南昌330008 [3]南昌大学第二附属医院康复科,南昌330008

出　　处：《中南大学学报（医学版）》2023年第1期24-33,共10页Journal of Central South University ：Medical Science

基　　金：江西省教育厅科技计划项目(GJJ200101);江西省卫生健康委员会科技计划项目(20203006)。

摘　　要：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(microtubuleassociated 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。Objective: 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.Conclusion: 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.

关 键 词：miR-18a-5p 心肌损伤 同型半胱氨酸 活性氧 自噬 NOTCH2 

分 类 号：R54[医药卫生—心血管疾病]