The role and regulatory mechanism of RNA binding protein ZFP36 in hypoxia/reoxygenation injury of cardiomyocytes
Objective To explore the role of ZFP36 in cardiomyocyte injury and autophagy induced by hypoxia/reoxygenation(H/R)so as to clarify its molecular regulatory mechanism.Methods H9C2 rat cardiomyocytes were infected with ZFP36 overexpressing lentivirus(OE-ZFP36)or its negative control lentivirus(OE-ZFP36 NC)to construct stable cell lines,respectively.Transfection of ATG4D overexpression plasmid(OE-ATG4D)improved the expression of ATG4D.Hypoxia/reoxygenation(H/R)induced myocardial cell injury.H9C2 cells were mainly divided into control group,H/R group,OE-ZFP36 NC+H/R group,OE-ZFP36+H/R group,OE-ATG4D NC+H/R group,OE-ATG4D+H/R group,OE-ZFP36+OE-ATG4D NC+H/R group,and OE-ZFP36+OE-ATG4D+H/R group.The protein expressions of ATG4D,Beclin1,LC3 and ZFP36 in H9C2 cells were detected by Western blotting.The mRNA levels of ZFP36 and ATG4D in H9C2 cells were detected by Real-time fluorescence quantitative PCR(qPCR).The viability of H9C2 cells was detected by CCK-8 assay.The levels of interleukin(IL-6)and tumor necrosis factor(TNF-α)in H9C2 cells were detected by enzyme-linked immunosorbent assay(ELISA).Reactive oxygen species(ROS)in H9C2 cells were detected by DCFH-DA method.SOD detection kit was used to detect the SOD level in H9C2 cells.The apoptosis of H9C2 cells was detected by flow cytometry.LC3 autophagosomes in H9C2 cells were detected by cellular immunofluorescence.Dual-luciferase reporter gene assay was used to detect the binding of ZFP36 and ATG4D mRNA in H9C2 cells.Results Compared with control group,H/R group showed decreased cell viability,increased IL-6 and TNF-α levels,increased ROS levels and decreased SOD levels,increased cell apoptosis.Up-regulated ATG4D and Beclin1 protein expression,increased LC3 Ⅱ/LC3 Ⅰ ratio,as well as upregulated ZFP36 expression were found in H/R group(all P＜0.05).Compared with OE-ZFP36 NC+H/R group,elevated cell viability,decreased IL-6 and TNF-α levels,decreased ROS levels and increased SOD levels,reduced cell apoptosis(P＜0.05),and downregulated ATG4D and Beclin1 protein expression,decreased LC3Ⅱ/LC3 Ⅰ ratio were shown in OE-ZFP36+H/R group(all P＜0.05).Compared with infection with OE-ZFP36 NC lentivirus,infection with OE-ZFP36 lentivirus decreased the luciferase activity of ATG4D 3'-UTR reporter gene,decreased the stability of ATG4D mRNA,and downregulated the H/R-induced ATG4D mRNA expression(all P＜0.05).Compared with OE-ATG4D NC+H/R group,OE-ATG4D+H/R group had upregulated ATG4D mRNA and protein expression,decreased cell viability,increased IL-6 and TNF-α levels,increased ROS levels,decreased SOD levels and elevated cell apoptosis(all P＜0.05).Compared with OE-ZFP36+OE-ATG4D NC+H/R group,OE-ZFP36+OE-ATG4D+H/R group had decreased cell viability,increased IL-6 and TNF-α levels,increased ROS levels,decreased SOD levels and elevated cell apoptosis(all P＜0.05).Conclusion The expression of ZFP36 is upregulated in H/R-induced cardiomyocyte injury.The overexpression of ZFP36 inhibits H/R-induced cardiomyocyte injury and autophagy by regulating ATG4D,thus resisting cardiomyocyte H/R injury.It proves that ZFP36 is an important regulatory molecule against MI/RI.
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