miR-219a-5p通过下调KLF4减轻H_(2)O_(2)诱导的人心肌细胞氧化损伤  

miR-219a-5p Inhibits H_(2)O_(2)-induced Oxidative Stress Injury in Human Cardiac Myocytes by Downregulating KLF4

作　　者：周云洁[1] 宋歌[2] 路永平[2] 韩丽华[3] Zhou Yunjie;Song Ge;Lu Yongping(Graduate Affairs Office,the Second Affiliated Hospital of Henan University of Chinese Medicine,Zhengzhou 450002,China;Department of Functional Examination,the Second Affiliated Hospital of Henan University of Chinese Medicine,Zhengzhou 450002,China)

机构地区：[1]河南中医药大学第二附属医院研究生办公室,郑州450002 [2]河南中医药大学第二附属医院功能检查科,郑州450002 [3]河南中医药大学第二附属医院心内科,郑州450002

出　　处：《华中科技大学学报（医学版）》2021年第6期723-727,共5页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：国家自然科学基金资助项目(No.81373610)。

摘　　要：目的探究miR-219a-5p对人心肌细胞(HCM)氧化应激损伤的作用和机制。方法利用H_(2)O_(2)建立HCM氧化应激模型,将HCM细胞随机分成4组:对照组、H_(2)O_(2)组(200μmol/L H_(2)O_(2)处理6 h),H_(2)O_(2)+NC mimic组(H_(2)O_(2)处理前转染对照mimic)和H_(2)O_(2)+miR-219a-5p组(H_(2)O_(2)处理前转染miR-219a-5p mimic)。噻唑蓝(MTT)法检测细胞活力,流式细胞术检测细胞凋亡,Real-time PCR检测miR-219a-5p和KLF4 mRNA表达,Western blot检测Bax、Bcl-2和Krüppel样因子4(KLF4)蛋白表达,试剂盒检测活性氧(ROS)、丙二醛(MDA)和超氧化物歧化酶(SOD)含量。另外,利用荧光素酶报告基因实验确证miR-219a-5p与KLF4的相互作用关系。结果与对照组相比,H_(2)O_(2)组中miR-219a-5p表达、细胞活力和SOD水平降低,细胞凋亡、Bax/Bcl-2、ROS和MDA水平以及KLF4表达升高(均P<0.05)。与H_(2)O_(2)组相比,H_(2)O_(2)+miR-219a-5p组中miR-219a-5p表达、细胞活力和SOD水平升高,细胞凋亡、Bax/Bcl-2、ROS和MDA水平以及KLF4表达降低(均P<0.05)。H_(2)O_(2)+NC mimic组与H_(2)O_(2)组相比上述指标变化均不大,差异均无统计学意义(均P>0.05)。荧光素酶实验结果显示,miR-219a-5p能够降低KLF4-野生型质粒的荧光素酶活性(P<0.05)。结论miR-219a-5p对H_(2)O_(2)诱导的HCM细胞氧化应激损伤具有缓解作用,这种作用可能是通过下调KLF4的表达发挥的。Objective To investigate the role and mechanism of miR-219 a-5 p on the oxidative stress injury of human cardiac myocytes(HCM).Methods Oxidative stress injury of HCM was established by H_(2)O_(2)stimulation.HCMs were randomly divided into 4 groups:control group,H_(2)O_(2)group(treatment with 200μmol/L H_(2)O_(2)for 6 h),H_(2)O_(2)+NC mimic group(transfected with normal control mimic before H_(2)O_(2)treatment),and H_(2)O_(2)+miR-219 a-5 p group(transfected with miR-219 a-5 p mimic before H_(2)O_(2)treatment).Cell viability and apoptosis were determined by MTT and flow cytometry,respectively.Real-time PCR was used to detect the mRNA expression of miR-219 a-5 p and KLF4.The protein levels of Bax,Bcl-2 and KLF4 were measured by Western blotting.The contents of reactive oxygen(ROS),malondialdehyde(MDA)and superoxide dismutase(SOD)were assayed by appropriate kits.Additionally,luciferase reporter system was used to confirm the relationship between miR-219 a-5 p and KLF4.Results Compared with the control group,miR-219 a-5 p expression,cell viability and SOD level were significantly decreased,whereas cell apoptosis,Bax/Bcl-2,and levels of ROS,MDA and KLF4 were remarkably increased in the H_(2)O_(2)group(P<0.05).Compared with the H_(2)O_(2)group,miR-219 a-5 p expression,cell viability and SOD level were notably elevated,but cell apoptosis,Bax/Bcl-2,and levels of ROS,MDA and KLF4 were dramatically reduced in the H_(2)O_(2)+miR-219 a-5 p group(P<0.05).However,these indexes were not changed in the H_(2)O_(2)+NC mimic group compared with the H_(2)O_(2)group(P>0.05).Luciferase reporter assay revealed that miR-219 a-5 p could reduce the luciferase activity of KLF4-wild type plasmid.Conclusion miR-219 a-5 p inhibits H_(2)O_(2)-induced oxidative stress injury of HCMs,which may be related to the inhibition of KLF4 expression.

关 键 词：miR-219a-5p KLF4 人心肌细胞 氧化应激损伤 

分 类 号：R34[医药卫生—基础医学]