摘要
目的探究miR-433-3p在过氧化氢(H_(2)O_(2))诱导的大鼠心肌细胞(H9c2)损伤中的作用及具体机制的研究。方法构建氧化应激损伤模型,以H9c2心肌细胞为研究对象,通过转染miR-433-3p模拟物(miR-433-3p mimics)、miRNA阴性对照(miR-NC)、阴性对照(pcDNA-NC)和MAPK8过表达质粒(pcDNA-MAPK8)至H_(2)O_(2)诱导的H9c2细胞中,将细胞分为对照组(Control),H_(2)O_(2)模型组(H_(2)O_(2)),H_(2)O_(2)+miRNA阴性对照组(H_(2)O_(2)+miR-NC),H_(2)O_(2)+miR-433-3p模拟物组(H_(2)O_(2)+miR-433-3p mimics),H_(2)O_(2)+miR-433-3p模拟物+pcDNA阴性对照组(H_(2)O_(2)+miR-433-3p mimics+pcDNA-NC)和H_(2)O_(2)+miR-433-3p模拟物+MAPK8过表达组(H_(2)O_(2)+miR-433-3p mimics+pcDNA-MAPK8)。采用qRT-PCR法检测miR-433-3p和MAPK8在H9c2细胞中的mRNA表达水平。MTT法和ELISA试剂盒分别检测细胞活性和乳酸脱氢酶(LDH)释放量。Western blot法检测Bax、Bcl-2、Caspase-3、Cleaved Caspase-3、MAPK8和GAPDH的蛋白表达水平。双荧光素酶报告实验检测miR-433-3p与MAPK8之间的靶向关系。结果与对照组相比,miR-433-3p在H_(2)O_(2)诱导的心肌细胞H9c2中低表达。相比较H_(2)O_(2)+miR-NC组,miR-433-3p过表达可以显著降低LDH释放量并增强细胞活力。此外,相比较H_(2)O_(2)+miR-NC组,miR-433-3p过表达可以显著降低促凋亡蛋白Bax和Cleaved Caspase-3的表达水平,而促进抗凋亡蛋白Bcl-2的表达水平。双荧光素酶报告实验显示miR-433-3p可以靶向结合MAPK8并负调控MAPK8的表达。过表达MAPK8可逆转miR-433-3p过表达对H_(2)O_(2)诱导的H9c2细胞活性损伤和细胞凋亡的抑制作用。结论miR-433-3p通过负靶向调控MAPK8在H_(2)O_(2)诱导的心肌细胞损伤中发挥保护作用。
Objective To explore the role and underlying mechanisms of miR-433-3p in hydrogen peroxide(H_(2)O_(2))-induced injury to H9c2 cardiomyocytes.Methods An oxidative stress injury model of H9c2 cardiomyocytes was established.H9c2 cardiomyocytes were transfected with miR-433-3p mimics,miRNA mimic negative control(miR-NC),pcDNA-NC,and a MAPK8 overexpression plasmid(pcDNA-MAPK8)and treated with H_(2)O_(2).H9c2 cells were divided into Control,H_(2)O_(2),H_(2)O_(2)+miR-NC,H_(2)O_(2)+miR-433-3p mimic,H_(2)O_(2)+miR-433-3p mimic+pcDNA-NC,and H_(2)O_(2)+miR-433-3p mimic+pcDNA-MAPK8 groups.The mRNA expressions of miR-433-3p and MAPK8 in H9c2 cells were detected by qRT-PCR assay.Cell viability and the amount of lactate dehydrogenase(LDH)released were detected by MTT assay and ELISA kits,respectively.Cell apoptotic-related protein expressions of Bax,Bcl-2,caspase-3,and cleaved caspase-3 were measured by Western blot analysis.The luciferase reporter assay was performed for testing the targeting relationship between miR-433-3p and MAPK8.Results Compared with the control group,the expression of miR-433-3p was lower in H_(2)O_(2)-induced H9c2 cardiomyocytes.Compared with the H_(2)O_(2)+miR-NC group,miR-433-3p overexpression significantly reduced the amount of LDH released and enhanced cell viability.In addition,compared with the H_(2)O_(2)+miR-NC group,miR-433-3p overexpression significantly decreased the expressions of the pro-apoptotic proteins Bax and cleaved caspase-3,but increased the expression of the anti-apoptotic protein Bcl-2.The luciferase reporter assay showed that miR-433-3p directly targeted MAPK8 and negatively regulated the expression of MAPK8.Overexpression of MAPK8 reversed the inhibitory effects of miR-433-3p overexpression in H_(2)O_(2)-induced H9c2 cell injury and apoptosis.Conclusions miR-433-3p has a protective role in cardiomyocyte injury induced by H_(2)O_(2)through the negative regulation of MAPK8.
作者
盛静宇
朱海根
王丽
SHENG Jingyu;ZHU Haigen;WANG Li(Department of Electrocardiography,Wujin Clinical College of Xuzhou Medical University,Wujin Hospital Affiliated to Jiangsu University,Changzhou 213000,China;Department of General Medicine,Wujin Clinical College of Xuzhou Medical University,Wujin Hospital Affiliated to Jiangsu University,Changzhou 213000;Department of Hospital-acquired Infection Control,Changzhou De’an Hospital,Changzhou 213000)
出处
《中国比较医学杂志》
CAS
北大核心
2021年第8期63-70,共8页
Chinese Journal of Comparative Medicine
基金
常州市科技应用基础研究计划资助项目(CJ20160001)。
