TP53BP2/ASPP2通过p53非依赖性方式激活mTOR通路抑制HepG2人肝癌细胞自噬

TP53BP2/ASPP2 inhibits autophagy of HepG2 cells by activating mTOR pathway in a p53-independent manner

目的研究肿瘤蛋白p53结合蛋白2/p53凋亡刺激蛋白2(TP53BP2/ASPP2)对HepG2人肝癌细胞自噬的调节作用和机制。方法通过腺病毒、慢病毒感染HepG2细胞上调或下调细胞中ASPP2的表达,HepG2细胞继续在不含胎牛血清的培养基中培养24 h,通过Western blot法检测自噬相关蛋白微管相关蛋白1轻链3(LC3)、beclin1、P62、自噬相关基因5(ATG5)、ATG7和哺乳动物雷帕霉素靶蛋白(mTOR)通路相关蛋白mTOR、磷酸化的mTOR(p-mTOR)、真核转录起始因子4E结合蛋白1(4EBP1)、磷酸化的4EBP1(p-4EBP1)、核糖体蛋白S6、磷酸化的S6(p-S6)、核糖体S6激酶B1(RPS6KB1/p70S6K)、磷酸化的p70S6K(p-p70S6K)的蛋白表达,荧光显微镜检测细胞表达的绿色荧光蛋白-微管相关蛋白1轻链3(GFP-LC3)融合蛋白。同时,使用慢病毒感染不表达p53的Hep3B细胞,敲低细胞中的ASPP2水平,Western blot法检测Hep3B细胞ASPP2和mTOR通路相关蛋白mTOR、p-mTOR、4EBP1、p-4EBP1、S6、p-S6、RPS6KB1/p70S6K、p-p70S6K的蛋白表达。结果当ASPP2的表达上调时,mTOR复合物1(mTORC1)通路被激活,并且自噬相关蛋白的表达和自噬体的数量减少。在下调ASPP2表达后,mTORC1通路受到抑制,自噬相关蛋白的表达水平和自噬体的数量增加。在p53表达沉默的Hep3B细胞中,当ASPP2下调后,mTORC1途径仍然受到抑制。结论ASPP2以p53非依赖性方式激活mTOR通路抑制HepG2人肝癌细胞自噬。

Objective To study the regulative effect of tumor protein p53 binding protein 2/p53 apoptosis-stimulating protein 2(TP53BP2/ASPP2)on the autophagy of HepG2 human hepatoma cells and its mechanism.Methods The expression of ASPP2 was up-regulated or down-regulated by HepG2 cells infected with adenovirus and lentivirus.HepG2 cells were cultured in medium without fetal bovine serum for 24 hours.Western blotting was used to detect the expression of microtubule-associated protein 1 light chain 3(LC3),beclin1,P62,autophagy-related gene 5(ATG5),ATG7,and mammalian target of rapamycin(mTOR)pathway-associated protein mTOR,phospho-mTOR(p-mTOR),eukaryotic transcription initiation factor 4E binding protein 1(4EBP1),phospho-4EBP1(p-4EBP1),ribosomal protein S6,phospho-S6(p-S6),ribosomal S6 kinase B1(RPS6KB1/p70S6K),phospho-p70S6K(p-p70S6K).Fluorescence microscopy was used to detect the green fluorescent protein-microtubule-associated protein 1 light chain 3(GFP-LC3)fusion protein expressed by the cells.At the same time,the Hep3B cells without p53 expression were infected with lentivirus,and the levels of ASPP2 in the cells were knocked down.Western blotting was performed to detect the expression of ASPP2 and mTOR pathway-associated protein mTOR,p-mTOR,4EBP1,p-4EBP1,S6,p-S6,RPS6KB1/p70S6K,p-p70S6K.Results When the expression of ASPP2 was up-regulated,the mTOR complex 1(mTORC1)pathway was activated,and the expression of autophagy-related proteins and the number of autophagosomes were reduced.After the expression of ASPP2 was down-regulated,the mTORC1 pathway was inhibited,and the expression level of autophagy-related proteins and the number of autophagosomes increased.In Hep3B cells with p53 expression silence,the mTORC1 pathway was still inhibited when ASPP2 was down-regulated.Conclusion ASPP2 inhibits the autophagy of HepG2 cells by activating mTOR pathway in a p53-independent manner.