干扰Nrf2基因表达对结直肠癌细胞功能和EMT通路的影响  

Effect of interference with Nrf2 gene expression on functionality and EMT pathway in colorectal cancer cells

作　　者：庄依泽 张杰 覃智杰 李飞[1] 罗淼[1] 范霞[1] 周洲[1] 黄璜[1] 唐应明[1] 郑春华 Zhuang Yize;Zhang Jie;Qin Zhijie;Li Fei;Luo Miao;Fan Xia;Zhou Zhou;Huang Huang;Tang Yingming;Zheng Chunhua(Department of Pediatric Surgery,Guizhou Provincial People's Hospital,Guiyang 550002,China;Department of Gastrointestinal Surgery,General Group 300 Hospital,Guiyang 550005,China)

机构地区：[1]贵州省人民医院小儿外科,贵阳550002 [2]通用医疗三〇〇医院胃肠外科,贵阳550005

出　　处：《肿瘤研究与临床》2024年第9期664-669,共6页Cancer Research and Clinic

基　　金：贵州省卫生健康委员会科学基金卫生项目(GZWKJ2021-177)。

摘　　要：目的探讨核因子E2相关因子2(Nrf2)基因体外对结直肠腺癌细胞增殖、凋亡的影响及其在调控上皮间质转化(EMT)通路中的作用。方法设计并合成3个Nrf2的小干扰RNA(siRNA)序列,分别为siRNA-223、siRNA-538、siRNA-756,并设计合成无关序列;构建载有Nrf2各siRNA序列的质粒,将载有siRNA序列质粒和载有无关序列质粒分别转染至人结直肠腺癌Caco-2细胞,分别为干扰组和空载组,另设未经任何处理的Caco-2细胞为对照组。采用实时荧光定量聚合酶链反应(RT-qPCR)法和蛋白质印迹(WB)法检测各组细胞中Nrf2基因转录水平和翻译水平相对表达量,以验证Nrf2干扰效果;选择干扰效果最好的siRNA进行后续实验。采用CCK-8法检测各组细胞增殖能力(以吸光度值表示);采用RT-qPCR法检测各组细胞EMT通路相关因子[波形蛋白(Vim)、N-钙黏蛋白(N-cad)及E-钙黏蛋白(E-cad)]转录水平相对表达量;采用WB法检测各组细胞中促凋亡蛋白Bax表达情况。结果RT-qPCR和WB法检测结果显示,siRNA-756干扰组Caco-2细胞中Nrf2基因转录水平和翻译水平相对表达量最低,与对照组、空载组比较,差异均有统计学意义(均P<0.05)。CCK-8法检测结果显示,对照组、空载组、siRNA-756干扰组Caco-2细胞培养48 h后的吸光度值分别为(100±5)%、(94±4)%、(82±5)%;与对照组和空载组相比,siRNA-756干扰组吸光度值均低,差异均有统计学意义(均P<0.05)。RT-qPCR法检测结果显示,siRNA-756干扰组Vim和N-cad转录水平相对表达量均高于对照组、空载组,差异均有统计学意义(均P<0.05);E-cad转录水平相对表达量均低于对照组、空载组,差异均有统计学意义(均P<0.05)。WB法检测结果显示,siRNA-756干扰组Bax蛋白相对表达量高于对照组,差异有统计学意义(P<0.05)。结论体外干扰Nrf2表达可减弱人结直肠腺癌Caco-2细胞增殖能力和抗凋亡能力,机制可能是Nrf2通过调控EMT通路的Vim、N-cad、E-cad表达来增强肿�ObjectiveTo investigate the effect of nuclear factor-erythroid 2-related factor 2(Nrf2)gene on the proliferation and apoptosis of colorectal adenocarcinoma cells in vitro,and the role of Nrf2 gene in regulation of epithelial-mesenchymal transition(EMT)pathway.MethodsThree Nrf2 small interfering RNA(siRNA)sequences were designed and synthesized,namely siRNA-223,siRNA-538 and siRNA-756,and the unrelated sequences were designed and synthesized.The plasmids carrying various siRNA sequences of Nrf2 were constructed,and the plasmids carrying siRNA sequences and the plasmids carrying unrelated sequences were transfected into human colorectal adenocarcinoma Caco-2 cells,namely interference group and empty vector group,respectively.Additionally,Caco-2 cells without any treatment were used as the control group.Real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)and Western blotting(WB)methods were used to detect the relative expression of Nrf2 gene in transcription and translation levels in each group of cells,in order to verify the interference effect of Nrf2;the siRNA with the best interference effect was selected for subsequent experiments.CCK-8 method was used to detect the proliferation ability of each group of cells(expressed as absorbance value);RT-qPCR was used to detect the relative expression of EMT pathway-related factors[vimentin(Vim),N-cadherin(N-cad)and E-cadherin(E-cad)]in transcription level in each group of cells;WB method was used to detect the expression of pro-apoptotic protein Bax in each group of cells.ResultsThe results of RT-qPCR and WB methods showed that compared with the control group and the empty group,the relative expression of Nrf2 gene in transcription and translation levels in Caco-2 cells of the siRNA-756 interference group were the lowest,and the differences were statistically significant(all P<0.05).The CCK-8 results showed that the absorbance values of Caco-2 cells in the control group,empty group and siRNA-756 interference group after 48 hours of culture were(100±5)%

关 键 词：结直肠肿瘤 核因子E2相关因子2 细胞凋亡 上皮间质转化 

分 类 号：R735.34[医药卫生—肿瘤]