SIRT3过表达通过调控细胞代谢转换抑制肾肿瘤细胞的增殖

SIRT3 Overexpression Inhibits Growth of Kidney Tumor Cells via Regulating Cellular Metabolic Transformation

目的:研究SIRT3对肾透明细胞癌(clear cell renal cell carcinoma, ccRCC)769-P细胞增殖和抗氧化能力的影响,并进一步探究其作用机制。方法:在769-P细胞的基础上构建SIRT3过表达稳转细胞系;利用CCK-8试剂检测769-P SIRT3过表达细胞的增殖速度;利用CellROX~Deep Red染料并结合流式细胞分析检测SIRT3过表达对769-P细胞中ROS水平的影响;利用定量蛋白质组学和代谢组学的方法,探究SIRT3对769-P细胞的作用机制。结果:CCK-8实验结果表明,769-P SIRT3过表达细胞的生长速度与对照细胞相比下降了约48%;定量蛋白质组学分析显示,769-P SIRT3过表达细胞中ALDOA、ALDOA、ENO2、PKM、LDHA、LDHB表达量下调约0.4至0.7倍,SDHB和CS上调约1.3倍;代谢组学分析显示,PEP、pyruvic acid、lactate、carnitine水平下降约0.4至0.7倍,isocitric acid和acetyl-CoA水平升高分别约1.3和2.8倍;分析还显示SIRT3过表达上调SOD2、TXN、GPX4和GLRX5的表达量约1.3至2倍,降低ROS水平约40%,增强细胞对过氧化氢的耐受力。结论:SIRT3过表达引起769-P细胞的代谢转换,从而抑制其增殖;且上调769-P细胞中抗氧化酶的表达,降低ROS水平,增强细胞的抗氧化能力。

Objective:To study the effect of SIRT3 on the proliferation and oxidation resistance of clear cell renal cell carcinoma(ccRCC)769-P cells,and to explore its mechanism.Methods:We established SIRT3 overexpressing stable cell line in 769-P cells.The CCK-8 assay was used to measure the proliferation rate of 769-P SIRT3 OE,while flow cytometry was used to determine the level of cellular reactive oxygen species in 769-P SIRT3 OE cells.To explore the mechanism of SIRT3 on 769-P cells by quantitative proteomics and metabonomics.Results:CCK-8 assay showed that SIRT3 overexpression decreased cell proliferation rate.Quantitative proteomics found that SIRT3 overexpression decreased the expression of ALDOA,ALDOA,ENO2,PKM,LDHA,LDHB,which increased the expression of SDHB and CS.Metabolomics found that SIRT3 overexpression decreased the level of PEP,pyruvic acid,lactate,carnitine,which increased the level of isocitric acid and acetyl-CoA.Furthermore,SIRT3 overexpression also increased the expression of SOD2,TXN,GPX4 and GLRX5,which decreased the level of cellular reactive oxygen species and enhanced resistance to oxidative stress.Conclusion:SIRT3 Overexpression inhibits growth of kidney tumor cells via regulating cellular metabolic transformation,while SIRT3 overexpression decreased the level of ROS and enhanced resistance to oxidative stress.