mTOR活化上调MGST1并增强细胞抗氧化能力

mTOR activation up-regulates MGST1 and enhances cellular antioxidant capacity

目的探讨雷帕霉素靶蛋白(mTOR)过度活化导致细胞抗氧化能力增强的分子机制。方法使用Gene Expression Omnibus(GEO)数据库中的芯片数据分析mTOR活化细胞与对照细胞的差异基因并进行功能通路富集分析。使用反应活性氧(ROS)诱导剂高剂量亚精胺处理细胞,观察mTOR高活化细胞TSC2敲除的小鼠胚胎成纤维细胞(tsc2^(-/-)MEFs)的抗氧化能力。利用差异表达基因之间的蛋白质相互作用筛选核心基因。使用q-PCR和Western blot验证该核心基因在tsc2^(-/-)MEFs中的表达情况。在tsc2^(-/-)MEFs中构建该核心基因的敲低细胞系并检测该基因对细胞增殖和抗氧化能力的影响。利用The Cancer Genome Atlas(TCGA)的RNA-seq和临床数据分析该基因对肝癌和黑色素瘤患者预后的影响。结果mTOR活化细胞中参与氧化还原通路的基因显著上调。mTOR活化的细胞对高剂量亚精胺更耐受(P<0.05)。分析筛选出该通路中差异表达的核心基因MGST1(P<0.05)。MGST1的表达在mTOR活化细胞中较对照细胞系显著增强(P<0.05)。在Rapamycin处理的mTOR活化细胞中,MGST1的表达水平下降。tsc2^(-/-)MEFs敲低MGST1后对氧化应激更敏感(P<0.05)。在TCGA的肝癌和黑色素瘤数据中发现,MGST1高表达的患者生存期低于低表达患者(P<0.01)。结论mTOR活化可以促进MGST1的表达使细胞的抗氧化能力增强。

Objective To investigate the molecular mechanism of enhanced antioxidant capacity in mammalian target of rapamycin(mTOR)over-activated cell.Methods Differential gene analysis,functional and pathway enrichment analysis in mTOR activated cell were conducted by using gene expression omnibus(GEO)database.The mTOR-hyperactive tsc2^(-/-)MEFs were treated with high dose spermidine to examine their antioxidant capacity.The core gene was screened by protein-protein interaction analysis.The expression of this core gene in mTOR activated cell was examined by q-PCR and Western blot.Core gene-knockdown cell line of mTOR activated cell was estab-lishedto study cell proliferation and antioxidant capacity.Core gene expression and prognosis of patient were analyzed in the TCGA liver cancer and melanoma data.Results The genes of the redox signaling in mTOR activated cells were significantly up-regulated.The mTOR activated cells were more resistant to high dose of spermidine(P<0.05).The core gene MGST1 was screened out due to its highest score of protein-protein interaction analysis(P<0.05).q-PCR and Western blot showed that MGST1 expression was significantly increased in mTOR activated cells(P<0.05).The expression of MGST1 decreased in rapamycin-treated mTOR activated cells.mTOR activated cell line was more sensitive to oxidative stress after MGST1 knockdown(P<0.05).Higher MGST1 expression was significantly associated with poor prognosis of patients based on TCGA liver cancer and melanoma data(P<0.05).Conclusions mTOR hyperactivation promotes the expression of MGST1 and enhances the antioxidant capacity of cells.