Methyltransferase WTAP aggravates hypoxia/reoxygenation-induced myocardial cell injury by regulating the expression of activator of transcription 4
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.Methods ① Experiment Ⅰ: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.② Experiment Ⅱ: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.③ Experiment Ⅲ: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.Results ①ExperimentⅠ: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.②Experiment Ⅱ: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.③ Experiment Ⅲ: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.
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