RNA结合蛋白ZFP36在心肌细胞缺氧/复氧损伤中的作用及调控机制

The role and regulatory mechanism of RNA binding protein ZFP36 in hypoxia/reoxygenation injury of cardiomyocytes

目的探讨RNA结合锌指蛋白36(ZFP36)在缺氧/复氧(H/R)诱导的心肌细胞损伤和自噬中的作用及分子调控机制。方法H9C2大鼠心肌细胞感染ZFP36过表达慢病毒(OE-ZFP36)或其阴性对照慢病毒(OE-ZFP36 NC)构建稳转细胞株;转染ATG4D过表达质粒(OE-ATG4D)提高ATG4D表达。H/R处理诱导心肌细胞损伤;将H9C2细胞分为对照组、H/R组、OE-ZFP36 NC+H/R组、OE-ZFP36+H/R组、OE-ATG4D NC+H/R组、OE-ATG4D+H/R组、OE-ZFP36+OE-ATG4D NC+H/R组和OE-ZFP36+OE-ATG4D+H/R组。各组细胞应用Western blotting检测ATG4D、Beclin1、LC3和ZFP36蛋白表达;实时荧光定量PCR(qPCR)检测ZFP36和ATG4D mRNA表达;CCK-8检测细胞活性;酶联免疫吸附法(ELISA)检测白介素6(IL-6)和肿瘤坏死因子(TNF-α)水平;DCFH-DA法检测活性氧(ROS)水平;SOD试剂盒检测SOD水平;流式细胞术检测细胞凋亡;细胞免疫荧光检测LC3自噬体;双荧光素酶报告基因实验检测ZFP36和ATG4D mRNA的结合。结果与对照组比较,H/R组中细胞活性降低,IL-6和TNF-α水平提高,ROS水平升高而SOD水平降低,细胞凋亡增加;ATG4D、Beclin1蛋白表达上调,同时LC3Ⅱ/LC3Ⅰ比值也显著增加;且ZFP36表达上调(P<0.05)。与OE-ZFP36 NC+H/R组比较,OE-ZFP36+H/R组中细胞活性提高,IL-6和TNF-α水平降低,ROS水平下降而SOD水平升高,细胞凋亡减少,且ATG4D、Beclin1蛋白表达下调,LC3Ⅱ/LC3Ⅰ比值也显著下调(P<0.05)。与感染OE-ZFP36 NC慢病毒比较,感染OE-ZFP36慢病毒降低ATG4D 3′-UTR报告基因的荧光素酶活性,降低ATG4D mRNA稳定性,下调H/R诱导的ATG4D mRNA表达(P<0.05)。与OE-ATG4D NC+H/R组相比,OE-ATG4D+H/R组中的ATG4D mRNA和蛋白表达显著上调,且细胞活性降低,IL-6和TNF-α水平增高,ROS水平升高而SOD水平降低,细胞凋亡增加(P<0.05)。与OE-ZFP36+OE-ATG4D NC+H/R组比较,OE-ZFP36+OE-ATG4D+H/R组中细胞活性降低,IL-6和TNF-α水平增高,ROS水平升高而SOD水平降低,细胞凋亡增加(P<0.05)。结论ZFP36在H/R诱导的心肌细胞损伤中表达上调,过表达ZFP36抑制了H/R诱导的心肌细胞损伤,并通过调控ATG4D抑制自噬,进而抵抗心肌细胞H/R损伤,证明ZFP36是抵抗心肌缺血再灌注损伤的重要调控分子。

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 LC3Ⅱ/LC3Ⅰ 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/LC3Ⅰ 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.