甲基化转移酶WTAP调节转录激活因子4表达加剧缺氧/复氧诱导的心肌细胞损伤  

Methyltransferase WTAP aggravates hypoxia/reoxygenation-induced myocardial cell injury by regulating the expression of activator of transcription 4

作　　者：杨娜[1] 曹敏[2] Yang Na;Cao Min(Department of Anesthesiology,the Third People's Hospital of Chengdu,Chengdu 610000,Sichuan,China;Department of Urology,the Third People's Hospital of Chengdu,Chengdu 610000,Sichuan,China)

机构地区：[1]成都市第三人民医院麻醉科,四川成都610000 [2]成都市第三人民医院泌尿外科,四川成都610000

出　　处：《中华危重病急救医学》2024年第3期279-285,共7页Chinese Critical Care Medicine

基　　金：四川省科技计划项目(2022YFS0307)。

摘　　要：目的观察甲基化转移酶Wilms肿瘤蛋白1相关蛋白(WTAP)在缺氧/复氧(H/R)诱导的心肌细胞损伤中的调节作用及其分子机制。方法①实验一:将H9C2心肌细胞分为空白对照组和H/R模型组。采用H/R诱导H9C2细胞建立心肌缺血/再灌注(I/R)损伤模型;空白对照组不进行处理。采用N6-甲基腺苷(m6A)RNA甲基化测定试剂盒检测m6A水平;分别采用实时荧光定量聚合酶链反应(RT-qPCR)和蛋白质免疫印迹试验(Western blotting)检测甲基化转移酶〔WTAP、甲基转移酶样蛋白(METTL3、METTL14)〕的基因和蛋白表达水平。②实验二:将H9C2心肌细胞分为空白对照组、H/R+sh-NC组、H/R+sh-WTAP组。H/R+sh-WTAP组转染sh-WTAP以敲降WTAP表达,其余各组制模方法同实验一。转染48 h后,采用流式细胞术检测细胞凋亡率;采用Western blotting检测WTAP、活化天冬氨酸特异性半胱氨酸蛋白酶3(caspase-3)、活化多聚二磷酸腺苷酸核糖聚合酶(PARP)、转录激活因子4(ATF4)、蛋白激酶RNA样内质网激酶(PERK)、磷酸化PERK(p-PERK)和CCAAT/增强子结合蛋白同源蛋白(CHOP)的蛋白表达水平;采用免疫荧光染色观察ATF4阳性表达情况。③实验三:将H9C2心肌细胞分为空白对照组、H/R+sh-NC组、H/R+sh-WTAP和H/R+sh-WTAP+ATF4组。H/R+sh-WTAP+ATF4组用过表达质粒ATF4转染H9C2心肌细胞,其余各组制模方法同实验二。采用流式细胞术检测细胞凋亡率;采用Western blotting检测ATF4、CHOP、活化caspase-3和活化PARP的蛋白表达水平。结果①实验一:H/R模型组m6A甲基化水平较空白对照组显著上调。RT-qPCR检测结果显示,H/R模型组METTL3、METTL14和WTAP基因表达水平均较空白对照组显著上调,以WTAP上调最显著;Western blotting检测结果呈相同趋势。提示甲基化转移酶WTAP的表达水平在H/R诱导的心肌细胞中显著上调。②实验二:H/R+sh-WTAP组细胞凋亡水平较H/R+sh-NC组明显减少〔(14.16±1.58)%比(24.51±2.38)%,P<0.05〕。Western bloObjective To investigate the regulatory role of Wilms tumor 1-associating protein(WTAP)in hypoxia/reoxygenation(H/R)-induced cardiomyocyte injury and its molecular mechanism.Methods①ExperimentⅠ:H9C2 cardiomyocytes were divided into blank control group and H/R model group.H/R was used to induce myocardial ischemia/reperfusion(I/R)injury model in H9C2 cells.The blank control group was not treated.N6-methyladenosine(m6A)RNA methylation assay kit was used to detect the level of m6A.Real-time fluorescent quantitative polymerase chain reaction(RT-qPCR)and Western blotting were used to detect the mRNA and protein expression levels of methyltransferases[WTAP,methyltransferase-like proteins(METTL3,METTL14)],respectively.②ExperimentⅡ:H9C2 cardiomyocytes were divided into blank control group,H/R+sh-NC group,and H/R+sh-WTAP group.sh-WTAP was transfected to knock down the expression of WTAP in H/R+sh-WTAP group,and the model establishment method in the other groups was the same as experimentⅠ.At 48 hours after transfection,the apoptosis rate of cells was detected by flow cytometry.The protein expressions of WTAP,activated caspase-3,activated poly(ADP-ribose)polymerase(PARP),activating transcription factor 4(ATF4),proline-rich receptor-like protein kinase(PERK),phosphorylated PERK(p-PERK)and CCAAT/enhancer-binding protein homologous protein(CHOP)were detected by Western blotting.The positive expression of ATF4 was observed by immunofluorescence staining.③ExperimentⅢ:H9C2 cardiomyocytes were divided into blank control group,H/R+sh-NC group,H/R+sh-WTAP group and H/R+sh-WTAP+ATF4 group.The overexpression plasmid ATF4 was transfected into H9C2 cardiomyocytes,and the modeling method of the other groups were modeled the same as experimentⅡ.The apoptosis rate was detected by flow cytometry.Western blotting was used to detect the protein expressions of ATF4,CHOP,activated caspase-3 and activated PARP.Results①ExperimentⅠ:the methylation level of m6A in the H/R group was significantly higher than that in the blank co

关 键 词：甲基转移酶 Wilms肿瘤蛋白1相关蛋白 细胞凋亡 内质网应激 缺氧/复氧 

分 类 号：R542.2[医药卫生—心血管疾病]