甘草酸通过RNA结合蛋白HuR调控p21表达对IEC-6细胞抗凋亡作用的影响

Glycyrrhizic Acid Protect IEC-6 Cells from Apoptosis Via HuR Mediated Posttranscription of p21

目的:研究甘草酸(GA)通过RNA结合蛋白人类抗原R(HuR)调控p21转录后表达,探讨GA对肿瘤坏死因子-α(TNF-α)/放线菌酮(CHX)诱导的小肠上皮细胞凋亡的抵抗作用机制。方法:采用大鼠小肠隐窝上皮细胞株(IEC-6),实验分空白组,GA(60μmol·L-1)组,TNF-α+CHX组和GA+TNF-α+CHX组,分别用生物素标记RNA与蛋白结合实验(Biotin pull down)和RNA-免疫共沉淀(RNA IP)分析RNA结合蛋白HuR与p21 mRNA的外源性和内源性结合;构建含p213’-UTR-ARE的荧光素酶报告质粒,用双荧光素酶报告基因系统检测HuR对p21的转录后调控影响;实时荧光定量聚合酶链式反应(Realtime PCR)检测HuR及p21 mRNA水平;蛋白免疫印迹法(Western blot)检测HuR及p21蛋白表达水平。采用TNF-α+CHX诱导细胞凋亡,实时细胞功能分析仪检测细胞凋亡,免疫共沉淀(CO-IP)检测p21蛋白与半胱氨酸蛋白酶-3前体蛋白(proCaspase-3)结合。结果:与空白组比较,GA处理后,能升高细胞浆内HuR蛋白的表达(P<0.05);GA能促进HuR与p21mRNA 3’-UTR的结合(P<0.05);GA通过HuR升高了p21 mRNA和蛋白表达水平(P<0.05),同时增高对荧光素酶报告基因的表达(P<0.01),沉默HuR后GA对p21 mRNA和蛋白的上调表达作用消失,GA能促进p21与proCaspase-3的结合(P<0.05),部分降低TNF-α+CHX诱导的IEC-6细胞凋亡。结论:GA能促进HuR向细胞浆内转位,促进HuR与p21 mRNA结合并调节其表达,增高荧光素酶表达活性以促进p21的翻译表达,增高p21与proCaspase-3的结合,从而提高IEC-6细胞的抗调亡能力。

Objective:To investigate the protective effect of glycyrrhizic acid(GA)from tumor necrosis factor-α+actinomycetes ketone(TNF-α+CHX)induced apoptosis in rat small intestine crypt epithelial cell line(IEC-6)via AU rich element mRNA binding protein HuR mediated posttranscription of p21 and the potential mechanism.Method:The cultured IEC-6 cells were observed.The experiment was divided into blank group,GA(60μmol·L-1)group,TNF-α+CHX group and GA+TNF-α+CHX group.Cytoplasmic and nuclear HuR were measured by Western blot.The interaction of HuR and p21 mRNA was detected by biotin pull down and RNA IP.Luciferase activity was measured after transfection with construct with p213’-UTR cloned into downstream of luciferase reporter.Cell apoptosis was detected by real-time dynamic cell analyzer,p21 and cysteine proteinas-3 precursor protein(proCaspase-3)association was analysised by CO-IP.Result:After GA treatment for 48 h,cytoplasmic HuR protein expression increased(P<0.05),the binding between HuR and p21 mRNA expression up regulated(P<0.05),luciferase activity increased(P<0.01),and p21 mRNA and protein expression also increased(P<0.05),while these results were abolished by HuR silencing with siRNA.GA enhanced p21 and procaspase3 interaction(P<0.05),and attenuated TNF-α+CHX induced apoptosis in IEC-6 cells.Conclusion:GA protected IEC-6 cells from TNF-α/CHX induced apoptosis via HuR mediated p21 posttranscription,which due to GA enhanced HuR binding to endogenous and recombinant p21 mRNA and increased p21 interaction with proCaspase3.