miR-499a-5p通过靶向TLR2抑制缺氧/复氧诱导的心肌细胞氧化应激和凋亡  

miR-499a-5p suppresses hypoxia/reoxygenation-induced cardiomyocyte oxidative stress and apoptosis by targeting TLR2

作　　者：杨伟[1] 王雁[1] 李俊杰[1] 王燕琼 陈文栋[1] YANG Wei;WANG Yan;LI Jun-jie;WANG Yan-qiong;CHEN Wen-dong(Department of Anesthesiology,the First Affiliated Hospital of Kunming Medical University,Kunming 650032,Yunnan,China)

机构地区：[1]昆明医科大学第一附属医院麻醉科,云南昆明650032

出　　处：《川北医学院学报》2022年第10期1239-1244,共6页Journal of North Sichuan Medical College

基　　金：云南省科技厅-昆明医科大学应用基础研究联合专项基金项目资助[2019FE001(-045)];云南省后备人才基金项目(H-2019028)。

摘　　要：目的:探究miR-499a-5p与TLR2的靶向关系,及其对缺氧/复氧(A/R)诱导的心肌细胞氧化应激和凋亡的影响。方法:培养H9c2细胞并分为模型组和对照组,对照组在常氧条件下培养,模型组为缺氧/复氧模型,并进一步分为miR-NC组、miR-499a-5p组、anti-miR-NC组、anti-miR-499a-5p组、miR-499a-5p+pGL3-Basic组和miR-499a-5p+pGL3-Basic+TLR2组;流式细胞仪检测各组细胞的凋亡率;qRT-PCR检测miR-499a-5p和TLR2 mRNA的表达水平;免疫印迹(Western blot)检测TLR2、B淋巴细胞瘤因子2XL(Bcl-xL)、Bcl-2相关X蛋白(Bax)、含半胱氨酸的天冬氨酸蛋白水解酶3(Caspase-3)蛋白表达;酶联免疫吸附法(ELISA)检测活性氧(ROS)、超氧化物歧化酶(SOD)、丙二醛(MDA)含量;双荧光素报告基因检测验证miR-499a-5p与TLR2的靶向关系。结果:与对照组相比,模型组中miR-499a-5p表达降低,TLR2表达升高(P<0.05);过表达miR-499a-5p可降低A/R模型心肌细胞凋亡和氧化应激作用,并靶向抑制TLR2;过表达TLR2逆转了miR-499a-5p对A/R模型心肌细胞抗凋亡及降低氧化应激的作用。结论:miR-499a-5p可能通过靶向抑制TLR2的激活,降低心肌细胞凋亡率及氧化应激反应,发挥保护心肌细胞的作用。Objective:To explore the targeting relationship between miR-499a-5p and TLR2,and its effect on hypoxia/reoxygenation-induced cardiomyocyte oxidative stress and apoptosis.Methods:H9c2 cells were cultured and grouped,the control group was cultured under normoxia,and the model group was made into a hypoxia/reoxygenation model.The model group was divided into miR-NC group,miR-499a-5p group,anti-miR-NC group,anti-miR-499a-5p group,miR-499a-5p+pGL3-Basic group,and miR-499a-5p+pGL3-Basic+TLR2 group.The apoptosis rate of each group was detected by flow cytometry,and the expression levels of miR-499a-5p and TLR2 mRNA were detected by QRT-PCR.The expression of TLR2,B-lymphocytoma factor 2XL(Bcl-XL),Bcl-2 associated X protein(Bax)and caspase-3 protein containing cysteine were detected by Western blot.The contents of reactive oxygen species(ROS),superoxide dismutase(SOD)and malondialdehyde(MDA)were determined by enzyme linked immunosorbent assay(ELISA).The targeting relationship between miR-499a-5p and TLR2 was verified by dual luciferin reporter gene assay.Results:Compared with the control group,the expression of miR-499a-5p in the model group was decreased,and the expression of TLR2 was increased,overexpression of miR-499a-5p could reduce the promotion of apoptosis and oxidative stress in A/R model cardiomyocytes,and targeted inhibition TLR2.Overexpression of TLR2 reversed the effects of miR-499a-5p on anti-apoptotic and oxidative stress reduction in A/R model cardiomyocytes.Conclusion:miR-499a-5p may play a role in protecting cardiomyocytes by inhibiting the activation of TLR2,reducing the apoptosis rate and oxidative stress response of cardiomyocytes.

关 键 词：miR-499 TLR2 心肌细胞 缺氧/复氧 氧化应激 细胞凋亡 

分 类 号：R542.2[医药卫生—心血管疾病]