沉默重组蛋白2对NCI-H1688细胞的抑制作用及其Wnt/β连环蛋白信号通路机制

Inhibitory effect of sileneing Frat2 on NCI-H1688 cells and its Wnt/βcatenin signaling pathway mechanism

目的:探讨T细胞淋巴瘤常见重排蛋白2(Frat2)通过Wnt/β连环蛋白(β-catenin)信号通路对小细胞肺癌NCI-H1688细胞增殖的影响,阐明其可能机制。方法:选取生长状态良好的人小细胞肺癌NCI-H1688细胞和正常支气管上皮样细胞HBE细胞,采用Western blotting法和逆转录实时荧光定量-聚合酶链反应(Real-Time RT-qPCR)法分别检测NCI-H1688细胞和HBE细胞中Frat2蛋白及mRNA表达水平。NCI-H1688细胞随机分为空白对照组、阴性对照组、Frat2-siRNA组和Frat2-siRNA+XAV组。空白对照组细胞不转染,阴性对照组细胞转染阴性对照慢病毒,Frat2-siRNA组转染Frat2-siRNA慢病毒,Frat2-siRNA+XAV组转染Frat2-siRNA慢病毒并同时加入4μmol·L^-1 Wnt信号通路抑制剂XAV939。采用Western blotting法和Real-Time RT-qPCR法分别检测转染后各组细胞中Frat2蛋白和mRNA表达水平,采用MTT法检测各组细胞增殖活性,采用流式细胞术检测不同细胞周期细胞百分率,采用Western blotting法检测细胞中增殖细胞核抗原(PCNA)、c-myc、细胞周期蛋白D1(Cyclin D1)、β-catenin和磷酸化糖原合成酶激酶3(pGSK-3β)蛋白表达水平。结果:与HBE细胞比较,NCI-H1688细胞中Frat2蛋白和mRNA表达水平明显升高(P<0.01)。与空白对照组和阴性对照组比较,Frat2-siRNA组NCI-H1688细胞中Frat2蛋白和mRNA表达水平明显升高(P<0.05);空白对照组与阴性对照组NCI-H1688细胞中Frat2蛋白和mRNA表达水平比较差异无统计学意义(P>0.05)。与空白对照组和阴性对照组比较,Frat2-siRNA组细胞增殖活性降低(P<0.05),G 0/G 1期细胞百分率升高(P<0.05),S期和G 2/M期细胞百分率降低(P<0.05),PCNA、c-myc、Cyclin D1、β-catenin和pGSK-3β蛋白表达水平降低(P<0.05);与Frat2-siRNA组比较,Frat2-siRNA+XAV组细胞增殖活性降低(P<0.05),G 0/G 1期细胞百分率升高(P<0.05),S期和G 2/M期细胞百分率降低(P<0.05),PCNA、c-myc、Cyclin D1、β-catenin和pGSK-3β蛋白表达水平降低(P<0.05);空白对照组与阴性对照组上述各指标比较差异无统计学意义(P>0.05)。结论:沉默Frat2可抑制小细胞肺癌细胞增殖,其机制可能与抑制Wnt/β-catenin信号通路有关。

Objective:To investigate the effect of frequently rearranged in advanced T-cell lymphomas 2(Frat2)on the proliferation of small cell lung cancer NCI-H1688 cells via Wnt/β-catenin signaling pathway,and to elucidate its possible mechanism.Methods:The human small cell lung cancer NCI-H1688 cells with good growth status and the normal bronchial epithelial-like cells HBE were selected;Western blotting method and Real-Ti me RT-qPCR were used to detect the expression levels of Frat2 mRNA and protein in the NCI-H1688 cells and the HBE cells.The NCI-H1688 cells were randomly divided into blank control group,negative control group,Frat2-siRNA group and Frat2-siRNA+XAV group.The cells in blank control group were not transfected.The cells in negative control group were transfected with negative control lentivirus,the cells in Frat2-siRNA group were transfected with Frat2-siRNA lentivirus and the cells in Frat2-siRNA+XAV group were transfected with the Frat2-siRNA lentivirus and treated with 4μmol·L^-1 of Wnt signaling pathway inhibitor XAV939.The expression levels of Frat2 protein and mRNA in the cells in various groups were determined by Western blotting method and Real-Time RT-qPCR method.The proliferation activities of the cells in various groups were determined by MTT method.The percentages of cells in different cell cycles were measured by flow cytometry.The expression levels of Frat2,proliferating cell nuclear antigen(PCNA),c-myc,cyclin D1,β-catenin and phosphorylated glycogen synthase kinase 3(pGSK-3β)proteins in the cells were determined by Western blotting method.Results:Compared with the normal bronchial epithelial-like cells HBE,the levels of Frat2 protein and mRNA in the NCI-H1688 cells were elevated(P<0.01).Compared with blank control group and negative control group,the expression levels of protein and Frat2 mRNA in Frat2-siRNA group were increased(P<0.05).There were no significant difference in the expression levels of Frat2 protein and mRNA in the NCI-H1688 cells between blank control group and negative control group(P>0.05).Compared with blank control group and negative control group,the proliferation activity of the cells in Frat2-siRNA group was decreased(P<0.05),the percentage of cells in G 0/G 1 phase was increased(P<0.05),the percentages of cells in S phase and G 2/M phase were decreased(P<0.05),and the expression levels of PCNA,c-myc,Cyclin D1,β-catenin and pGSK-3βproteins were decreased(P<0.05).Compared with Frat2-siRNA group,the proliferation activity of cells in Frat2 mRNA+XAV group was decreased(P<0.05),the percentage of cells in G 0/G 1 phase was increased(P<0.05),the percentages of cells in S phase and G 2/M phase were decreased(P<0.05),and the expression levels of PCNA,c-myc,Cyclin D1,β-catenin and pGSK-3βproteins were decreased(P<0.05).There were no significant differences in the indexes mentioned above between blank control group and negative control group(P>0.05).Conclusion:Silencing Frat2 can inhibit the proliferation of small cell lung cancer cells,and its mechanism may be related to the inhibition of Wnt/β-catenin signaling pathway.