敲减TLR2基因对结直肠癌细胞增殖的抑制作用及其机制

Inhibitory effect of knockdown of TLR2 gene on proliferation of colorectal cancer cells and its mechanism

目的:应用慢病毒RNA干扰技术敲减结直肠癌细胞中Toll样受体2(TLR2)基因,探讨其对结直肠癌细胞增殖的作用,并阐明其作用机制。方法:将处于对数生长期的结直肠癌HCT116细胞和HT29细胞分为未感染对照组、阴性对照组及基因敲减组(TLR2-RNAi组),分别给予无慢病毒感染、慢病毒RNAi及慢病毒TLR2-RNAi稳定转染。感染后采用蛋白印迹法检测各组细胞中TLR2蛋白表达水平,采用CCK-8法和流式细胞术检测各组细胞增殖活性及不同细胞周期细胞百分率,蛋白印迹法检测各组细胞中磷脂酰肌醇3-激酶/蛋白激酶(PI3K/Akt)和核因子κB(NF-κB)细胞信号通路相关蛋白及细胞周期相关蛋白cyclin D1、cyclin D3蛋白表达水平。结果:与未感染对照组和阴性对照组比较,TLR2-RNAi组HCT116细胞和HT29细胞中TLR2蛋白表达水平明显降低(P<0.01)。与未感染对照组和阴性对照组比较,TLR2-RNAi组HCT116细胞和HT29细胞增殖活性明显降低(P<0.05),S期和G 2期细胞百分率明显降低(P<0.05),G 1期细胞百分率明显升高(P<0.05)。与未感染对照组和阴性对照组比较,TLR2-RNAi组HCT116细胞和HT29细胞中PI3K、p-Akt、p-NF-κβ、cyclin D1和cyclin D3蛋白表达水平明显降低(P<0.01)。结论:敲减TLR2基因可以通过调控PI3K/Akt和NF-κB细胞信号通路及细胞周期相关蛋白cyclin D1和cyclin D3的表达而抑制结直肠癌细胞的增殖。

Objective:To knock down the Toll-like receptor 2(TLR2)gene in the colorectal cancer cells with lentiviral RNA interference technology,to explore its effect on the colorectal cancer cell proliferation,and to clarify its mechanism.Methods:The colorectal cancer HCT116 cells and HT29 cells in logarithmic phase were divided into non-infection control group,negative control group and gene knockdown group(TLR2-RNAi group);the cells were transfected with lentivirus-free,lentivirus-RNAi and lentivirus-TLR2-RNAi,respectively.The expression levels of TLR2 protein in the cells in various groups were detected by Western blotting method after stable transfection.The cell proliferation activities and the percentages of cells in different cell cycles were detected by CCK-8 method and flow cytometry.Western blotting method was used to detect the expression levels of PI3K/Akt and nuclear factor-κB(NF-κB)signaling pathway-related proteins and cell cycle-associated proteins cyclin D1 and cyclin D3 proteins in the cells in various groups.Results:Compared with non-infection control group and negative control group,the expression levels of TLR2 protein in the HCT116 cells and the HT29 cells in TLR2-RNAi group were decreased significantly(P<0.01).Compared with non-infection control group and negative control group,the cell proliferation activities in the HCT116 cells and the HT29 cells in TLR2-RNAi group were significantly decreased(P<0.05);the percentages of cells in S phase and G 2 phase were decreased significantly(P<0.05),and the percentages of cells in G 1 phase were increased significantly(P<0.05).Compared with non-infection control group and negative control group,the expression levels of PI3K,p-Akt,p-NF-κB,cyclin D1 and cyclin D3 proteins in the HCT116 cells and HT29 cells in TLR2-RNAi group were significantly decreased(P<0.01).Conclusion:Knockdown of TLR2 gene can inhibit the proliferation of colorectal cancer cells by regulating the expressions of PI3K/Akt and NF-κB cell signaling pathway proteins and cell cycle-associated proteins.