沉默信息调节因子2对大肠癌细胞有氧糖酵解和生长增殖的调控作用及其机制

Regulatory effect of silent information regulator 2 on aerobic glycolysis and growth and proliferation of colorectal cancer cells and its mechanism

目的:探讨沉默信息调节因子2(SIRT2)对大肠癌细胞有氧糖酵解和生长增殖的影响,阐明其相关作用机制。方法:选择大肠癌HCT116细胞和SW480细胞进行培养,采用Western blotting法检测2种细胞中SIRT2蛋白表达情况。选择SIRT2蛋白表达高的大肠癌HCT116细胞进行后续RNA干扰实验,设立阴性对照组、SIRT2-siRNA#1组、SIRT2-siRNA#2组和SIRT2-siRNA#3组;选择SIRT2蛋白表达低的大肠癌SW480细胞进行后续过表达实验,构建SIRT2重组质粒,选取处于对数生长期的SW480细胞,设立空白组、空载体质粒转染组和SIRT2质粒转染组;采用HCT116细胞进行回复实验,设立对照组、SIRT2-siRNA和葡萄糖-6磷酸脱氢酶(G6PD)质粒共转染组和SIRT2-siRNA#3组。利用Lipofectamine2000将质粒和siRNA分别转染至SW480细胞和HCT116细胞,验证沉默和过表达SIRT2后,采用葡萄糖氧化酶法检测培养液中葡萄糖水平,比色法检测培养液中乳酸水平,克隆形成实验检测细胞克隆形成率,CCK-8实验检测细胞增殖活性。反转录PCR(RTPCR)法检测沉默和过表达SIRT2后细胞中G6PD mRNA表达水平,Western blotting法检测沉默和过表达SIRT2后细胞中G6PD蛋白表达水平。免疫组织化学法检测大肠癌组织中SIRT2和G6PD蛋白表达水平。共转染SIRT2-siRNA和G6PD质粒后检测HCT116细胞中葡萄糖水平、乳酸水平、克隆形成率和细胞增殖活性。结果:与阴性对照组比较,SIRT2-siRNA#3组培养液中葡萄糖水平明显升高(P<0.05),乳酸水平明显降低(P<0.05),细胞增殖活性和细胞克隆形成率明显降低(P<0.05),G6PD mRNA和蛋白表达水平降低(P<0.05);与空载体质粒转染组比较,SIRT2质粒转染组培养液中葡萄糖水平明显降低(P<0.05),乳酸水平明显升高(P<0.05),细胞增殖活性和克隆形成率明显升高(P<0.05),G6PD mRNA和蛋白表达水平升高(P<0.05)。在大肠癌组织中SIRT2和G6PD蛋白表达水平均明显高于癌旁组织(P<0.05)。与SIRT2-siRNA#3组比较,SIRT2-siRNA和G6PD质粒共转染组培养液中的葡萄糖水平明显降低(P<0.05),乳酸水平明显升高(P<0.05),细胞增殖活性和克隆形成率明显升高(P<0.05)。结论:SIRT2促进大肠癌细胞有氧糖酵解及细胞生长增殖,其机制可能与SIRT2调控G6PD基因表达有关。

Objective:To investigate the effect of silent information regulator 2(SIRT2)on the aerobic glycolysis,growth and proliferation of the colorectal cancer cells,and to clarify its related mechanism.Methods:The HCT116 cells and SW480 cells were cultured,and Western blotting method was used to detect the expression levels of SIRT2 protein in two kinds of cells.The HCT116 cells with high expression of SIRT2 protein were selected for the following RNA interference experiment.Negative control group,SIRT2-siRNA#1 group,SIRT2-siRNA#2 group,and SIRT2-siRNA#3 group were established;the SW480 cells with low expression of SIRT2 protein were selected for the following SIRT2 overexpression experiment.The SIRT2 recombinant plasmids were constructed,and the SW480 cells at logarithmic growth phase were selected;blank group,empty vector plasmid transfection group,and SIRT2 plasmid transfection group were established;the recovery experiment was carried out in the HCT116 cells;control group,SIRT2-siRNA+glucose-6-phosphate dehydrogense(G6PD)plasmid co-transfection group and SIRT2-siRNA#3 group were established.The plasmid and siRNA were transfected into the SW480 cells and HCT116 cells by Lipofectamine 2000,respectively,the conterts of glucose in the culture medium in various groups were measured by glucose oxidase method after silencing SIRT2 and over-expression of SIRT2;the levels of lactic acid in the culture medium in various groups were measured by colorimetry after silencing SIRT2 and over-expression of SIRT2;the clone formation rates of the cells in various groups were determined by clonal formation assay;the proliferation activities of the cells in various groups were determined by CCK-8 assay;the expression levels of G6PD mRNA in the cells after silencing SIRT2 and over-expression of SIRT2 were determined by reverse transcriptionPCR(RT-PCR)method;the expression levels of G6PD protein in the cells in various groups after silencing SIRT2 and over-expression of SIRT2 were detected by Western blotting method;the expression levels of SIRT2 and G6PD proteins in colorectal cancer tissue were determined by immunohistochemistry.After co-transfection of SIRT2-siRNA and G6PD plasmids,the glucose levels,lactic acid levels,survival rates,and proliferation abilities of the cells were detected.Results:Compared with negative control group,the level of glucose in the culture medium in SIRT2-siRNA#3 group was increased significantly(P<0.05),the level of lactic acid was significantly decreased(P<0.05),the survival rate and proliferation ability of the cells were significantly decreased(P<0.05),the expression levels of G6PD mRNA and protein were decreased(P<0.05);compared with empty vector plasmid transfection group,the level of glucose in the culture medium in SIRT2 plasmid transfection group was decreased significantly(P<0.05),the level of lactic acid was significantly increased(P<0.05),the proliferation ability and clone formation rate of the cells were significantly increased(P<0.05).The expression levels of SIRT2 and G6PD proteins in colorectal cancer tissue were significantly higher than that in paracancerous tissue(P<0.05).Compared with SIRT2-siRNA#3 group,the level of glucose in the culture medium in SIRT2-siRNA+G6PD plasmid co-transfection group was significantly decreased(P<0.05),the level of lactic acid in the culture medium was significantly increased(P<0.05),and the proliferation ability and clone formation rate of the cells were significantly increased(P<0.05).Conclusion:SIRT2 promotes the aerobic glycolysis and growth and proliferation of the colorectal cancer cells,and its mechanism may be related to the expression of G6PD regulated by SIRT2.