The Research On The Effect And Mechanism Of PNPT1 In Ischemic Myocardial Injury

Objectives:1.To explore the role of apoptosis-related mRNA in hypoxic injury of cardiomyocytes.2.To explore the effects of hypoxic stimulation on the expression and localization of Polyribonucleotide nucleotidyltransferase 1(PNPT1)in cardiomyocytes.3.To explore the effect of knockdown PNPT1 on hypoxic myocardial injury and apoptosis-related mRNA in cardiomyocytes.4.To explore the role of Poly(A)Binding Protein Cytoplasmic 1(PABPC1)in PNPT1/ apoptosis-related mRNA degradation-mediated hypoxic injury of cardiomyocytes.Methods:1.To investigate the role of mRNA in hypoxic injury of cardiomyocytes.We cultured cardiomyocytes in an anaerobic environment to mimic myocardial ischemia.First of all,we divided the cardiomyocytes into 4 groups,treated them with hypoxia of 0h,4h,8h,and 12 h respectively,detected cell viability by CCK-8 method,screened out the time points with obvious apoptosis,and then used the FITC-PI kit to detect the apoptosis rate of cardiomyocytes to prove that the hypoxic cardiomyocyte model was successfully constructed;then we detected the apoptosis-related mRNA(ACTB mRNA,TUBA mRNA,SDHA mRNA,GAPDH mRNA)of the Control group and the Hypoxia group by q RT-PCR.Different concentration gradients(0n M,200 n M,400 n M,600 n M,800 n M,1000 n M)were set to screen the optimal concentration of Staurosporine(STS)induced cardiomyocyte apoptosis,and finally the apoptosis rate of STS group and Vehicle was detected with the FITC-PI kit,the content of apoptosis-related mRNA was detected by q RT-PCR.2.To explore the effects of hypoxic stimulation on the expression and localization of PNPT1 in cardiomyocytes.First of all,we divided the cardiomyocytes into 4 groups,extracted their mitochondrial proteins and cytoplasmic proteins after hypoxia treatment of 0h,4h,8h and 12 h respectively,and detected the content of PNPT1 protein by western blot to screen out the most obvious time point;then divided into Control group and Hypoxia group,and observed the changes of PNPT1 and mitochondrial co-localization under confocal microscopy by cellular immunofluorescence staining.3.To explore the effect of knockdown PNPT1 on hypoxic myocardial injury and mRNA degradation in cardiomyocytes.We first divided the cells into NC-si RNA group,PNPT1-si RNA1 group,PNPT1-si RNA2 group and PNPT1-si RNA3 group,and detected the content of PNPT1 protein by western blot,the mRNA level of PNPT1 by q RT-PCR to screen out the optimal sequence and further verified knockdown efficiency;then divided the cells into 4groups: si-Control group,si-PNPT1 group,si-Control+Hypoxia group and siPNPT1+Hypoxia group,and then detected PNPT1 content by western blot,used the light microscope to observe the changes in the growth state of each group,JC-1 kit to detect changes in mitochondrial membrane potential,transmission electron microscopy to observe changes in mitochondrial morphology,FITC-PI kit to detect apoptosis rate,q RT-PCR to detect the content of apoptosis-related mRNA.4.To explore the role of PABPC1 in PNPT1/ mRNA degradation-mediated hypoxic injury of cardiomyocytes.We first divided the cardiomyocytes into 4 groups,treated them with hypoxia at 0h,4h,8h and 12 h respectively,then detected the protein content of PABPC1 by western blot to screen out the most obvious time point;then constructed PNPT1 and PABPC1 overexpression cardiomyocytes,and detected the protein expression of PNPT1 and PABPC1 by western blot respectively to verify overexpression success.Then PNPT1 stable overexpression cardiomyocytes were divided into Pc-control +Hypoxia group and PcPABPC1+Hypoxia group.Finally,the changes in mitochondrial morphology were observed by transmission electron microscopy,the apoptosis rate was detected by FITC-PI kit,and the content of apoptosis-related mRNA was detected by q RT-PCR.Results:1.With the prolongation of hypoxia time,the activity of cardiomyocytes presented a gradual decreasing trend,and the apoptosis of hypoxic 12 hours is obvious,indicating that the model of hypoxic cardiomyocyte damage is successfully constructed.2.Under the stimulation of hypoxia,the degradation of apoptosis-related mRNA in cardiomyocytes increased.And after STS inducing apoptosis in cardiomyocytes,the degradation of apoptosis-related mRNA increased.3.With the prolongation of hypoxia time,the content of PNPT1 in cardiomyocyte mitochondria showed an increasing trend and a decreasing trend in the cytoplasm;after 12 hours stimulation of hypoxia,the co-localization of PNPT1 and mitochondrial in cardiomyocytes was significantly worse than that of the control group,and PNPT1 had a tendency to leak from the mitochondria.4.The PNPT1 protein level of the PNPT1-si RNA1 group and PNPT1-si RNA2 group was significantly reduced compared with the NC-si RNA group,the protein and mRNA level of PNPT1 in the si-PNPT1 group were significantly reduced compared with the si-Control group,indicating the successful knockdown of PNPT1 expression in cardiomyocytes;compared with the si-Control+Hypoxia group,the content of cytoplasmic PNPT1 protein in cardiomyocytes in the si-PNPT1+Hypoxia group was significantly reduced,the cell state was significantly improved,the mitochondrial membrane potential was restored,the mitochondrial morphology was more complete,the apoptosis rate was significantly reduced,and the apoptosis-related mRNA degradation was reduced.5.With the prolongation of hypoxia,the content of PABPC1 in cardiomyocytes presented a decreasing trend,suggesting that PABPC1 may be involved in the injury process of hypoxic cardiomyocytes;the level of PNPT1 in Lv-PNPT1 group increased significantly compared with that of Lv-control group,indicating the successful construction of overexpressed PNPT1cardiomyocytes;and the content of PABPC1 protein in the Pc-PABPC1 group increased significantly compared with that of Pc-control group,indicating the successful construction of overexpressed PABPC1 cardiomyocytes.Compared with the Pc-control+Hypoxia group,the mitochondrial morphology of the cells in the Pc-PABPC1+Hypoxia group was improved,the apoptosis rate decreased significantly,and the apoptosis-related mRNA degradation decreased significantly,indicating that PABPC1 may be a protective factor for hypoxic cardiomyocyte injury mediated by PNPT1/ mRNA degradation.Conclusion:mRNA degradation is the intrinsic mechanism of apoptosis in hypoxic cardiomyocytes,and PNPT1 mitochondrial leakage is a necessary condition for its degradation activation.In addition,PABPC1 may alleviate hypoxic damage of cardiomyocytes mediated by PNPT1/mRNA degradation.