1、第 49 卷 第 2 期2023年 3 月吉林大学学报（医学版）Journal of Jilin University（Medicine Edition）Vol.49 No.2Mar.2023DOI：10.13481/j.1671587X.20230215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岳金金,庞一心,张秀梅（锦州医科大学基础医学院生物化学与分子生物学教研室,辽宁 锦州 121001）摘要 目的目的：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。方法方法：选择大肠癌 HCT116细胞和 SW480细胞进行
2、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
3、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（RT-PCR）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
4、-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（P0.05），乳酸水平明显降低（P0.05），细胞增殖活性和细胞克隆形成率明显降低（P0.05），G6PD mRNA 和蛋白表达水平降低（P0.05）；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（P0.05），乳酸水平明显升高（P0.05），细胞增殖活性和克隆形成率明显升高（P0.05），G6PD mRNA 和蛋白表达水平升高（P0.05）。在大肠癌组织
5、中 SIRT2和 G6PD蛋白表达水平均明显高于癌旁组织（P0.05）。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（P0.05），乳酸水平明显升高（P0.05），细胞增殖活性和克隆形成率明显升高（P0.05）。结论结论：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。关键词 大肠肿瘤；沉默信息调节因子 2；葡萄糖-6磷酸脱氢酶；有氧糖酵解；生长增殖中图分类号 R735.3文献标志码 ARegulatory effect of silent information r
6、egulator 2 on aerobic glycolysis and growth and proliferation of colorectal cancer cells and its mechanismYUE Jinjin,PANG Yixin,ZHANG Xiumei文章编号 1671587X(2023)02038510收稿日期 20220602基金项目 辽宁省教育厅基础研究项目（JYTJCZR201910）作者简介 岳金金（1993），女，辽宁省阜新市人，讲师，理学硕士，主要从事肿瘤分子生物学方面的研究。通信作者 张秀梅，副教授，硕士研究生导师（E-mail：）385第 49 卷
7、 第 2 期 2023 年 3 月吉林大学学报（医学版）（Department of Biochemistry and Molecular Biology，School of Basic Medical Sciences，Jinzhou Medical University，Jinzhou 121001，China）ABSTRACT Objective：To investigate the effect of silent information regulator 2（SIRT2）on the aerobic glycolysis，growth and proliferation of th
8、e 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 t
9、he 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 co
10、nstructed，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（G6P
11、D）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 silen
12、cing 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 proliferati
13、on 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 group
14、s 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 lev
15、els，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（P0.05），the level of lactic acid was significantly decreased（P0.05），the survival rate an
16、d proliferation ability of the cells were significantly decreased（P0.05），the expression levels of G6PD mRNA and protein were decreased（P0.05）；compared with empty vector plasmid transfection group，the level of glucose in the culture medium in SIRT2 plasmid transfection group was decreased significantly（P0.05），the level of lactic acid was significantly increased（P0.05），the proliferation ability and clone formation rate of the cells were significantly increased（P0.05）.The expression levels of SIRT2