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

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 blotting检测结果显示,H/R+sh-WTAP组WTAP、活化caspase-3、活化PARP、p-PERK、ATF4和CHOP的蛋白表达水平均较H/R+sh-NC组显著下调。荧光显微镜下显示,H/R+sh-WTAP组ATF4阳性信号较H/R+sh-NC组显著减弱〔(19.36±1.81)%比(32.83±2.69)%,P<0.01〕。提示敲降WTAP可抑制H/R诱导的心肌细胞凋亡和内质网应激。③实验三:H/R+sh-WTAP+ATF4组细胞凋亡水平较H/R+sh-WTAP组显著增加〔(26.61±2.76)%比(17.14±0.87)%,P<0.05〕。Western blotting检测结果显示,H/R+sh-WTAP+ATF4组ATF4、CHOP、活化caspase-3和活化PARP的蛋白表达水平较H/R+sh-WTAP组显著上调。提示过表达ATF4可逆转sh-WTAP对H/R诱导的心肌细胞中内质网应激和细胞凋亡的抑制作用。结论甲基化转移酶WTAP可调节ATF4表达,介导细胞凋亡和内质网应激,促进H/R诱导的心肌细胞损伤。

Objective 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 control group.RT-qPCR results showed that the gene expressions of METTL3,METTL14 and WTAP in the H/R model group were significantly higher than those in the blank control group,and WTAP was the most significantly up-regulated.Western blotting results showed the same trend.These results suggested that the expression level of methyltransferase WTAP is significantly up-regulated in H/R-induced cardiomyocytes.②ExperimentⅡ:the apoptosis level in H/R+sh-WTAP group was significantly lower than that in H/R+sh-NC group[(14.16±1.58)%vs.(24.51±2.38)%,P<0.05].Western blotting results showed that the protein expressions of WTAP,activated caspase-3,activated PARP,p-PERK,ATF4 and CHOP in the H/R+sh-WTAP group were significantly lower than those in the H/R+sh-NC group.Fluorescence microscopy results showed that the ATF4 positive signal in the H/R+sh-WTAP group was significantly weaker than that in the H/R+sh-NC group[(19.36±1.81)%vs.(32.83±2.69)%,P<0.01].The above results suggested that knockdown of WTAP could inhibit H/R-induced cardiomyocyte apoptosis and endoplasmic reticulum stress.③ExperimentⅢ:the apoptosis level of H/R+sh-WTAP+ATF4 group was significantly higher than that of H/R+sh-WTAP group[(26.61±2.76)%vs.(17.14±0.87)%,P<0.05].Western blotting results showed that the protein expressions of ATF4,CHOP,activated caspase-3 and activated PARP in the H/R+sh-WTAP+ATF4 group were significantly higher than those in the H/R+sh-WTAP group.These results suggested that overexpression of ATF4 reversed the inhibitory effect of sh-WTAP on endoplasmic reticulum stress and apoptosis in H/R-induced cardiomyocytes.Conclusion Methyltransferase WTAP could regulate ATF4 expression,mediate cell apoptosis and endoplasmic reticulum stress,and promote H/R-induced myocardial cell injury.