Twist基因在肝细胞癌中的表达水平及其对细胞增殖、侵袭和上皮间质转化的影响

Twist gene expression level in hepatocellular carcinoma and its effect on cell proliferation,invasion and epithelial-mesenchymal transition

目的:检测肝细胞癌(HCC)中Twist的表达水平,并分析其对HCC细胞增殖、侵袭和上皮间质转化(EMT)的影响。方法:qRT-PCR检测HCC组织和细胞中Twist mRNA相对表达水平,Western blotting实验检测HCC细胞中Twist蛋白表达水平。si-Twist质粒转染至HepG2细胞中为实验组,同时设置阴性对照组(转染si-NC质粒)和对照组(无转染),qRT-PCR和Western blotting实验检测转染效率。MTT和集落形成实验、Transwell实验、Western blot实验分别检测Twist敲低后对细胞增殖、侵袭和EMT相关蛋白表达的影响。结果:HCC组织和细胞中Twist mRNA和蛋白表达水平增加;实验组Twist的2^(-ΔΔCt)值为0.32±0.08,明显低于对照组(1.00±0.10)和阴性对照组(1.02±0.09),差异有统计学意义(P<0.001)。实验组Twist蛋白灰度值为0.24±0.11,明显低于对照组(1.00±0.07)和阴性对照组(0.98±0.12),差异有统计学意义(P<0.001);实验组72 h的OD值为0.72±0.06,显著低于对照组(1.20±0.10)和阴性对照组(1.27±0.08),差异有统计学意义(P<0.001)。实验组形成的集落细胞数16.33±2.52显著低于对照组(44.67±4.51)和阴性对照组(47.12±4.05),差异有统计学意义(P<0.001);实验组发生侵袭的细胞数为54.33±9.50,低于对照组(102.14±12.50)和阴性对照组(105.62±14.35),差异有统计学意义(P<0.001);与对照组和阴性对照组相比,实验组E-钙黏着蛋白(E-cadherin)表达水平增加,波形蛋白(Vimentin)和N-钙黏着蛋白(N-cadherin)的表达水平降低。结论:Twist在肝癌组织中的表达水平增加,敲低其表达水平后可抑制细胞的增殖、侵袭和EMT。

Objective:Detect the expression level of Twist in hepatocellular carcinoma(HCC),and analyze its effect on HCC cell proliferation,invasion and epithelial-mesenchymal transition(EMT).Methods:qRT-PCR was used to detect the relative expression level of Twist mRNA in liver cancer tissues and cells,and Western blot was used to detect the expression level of Twist protein in liver cancer cells.The si-Twist plasmid was transfected into HepG2 cells as the experimental group.A negative control group(transfected with si-NC plasmid)and a control group(without transfection)were set at the same time,and the transfection efficiency was tested by qRT-PCR and Western blot experiments.MTT and colony formation experiments,transwell experiments,and Western blotting experiments were used to detect the effects of Twist knockdown on cell proliferation,invasion and EMT-related protein expression.Results:The expression levels of Twist mRNA and protein in liver cancer tissues and cells increased;The 2^(-ΔΔCt) value of Twist in the experimental group was 0.32±0.08,which was significantly lower than the control group 1.00±0.10 and the negative control group 1.02±0.09(P<0.001).The gray value of Twist protein in the experimental group was 0.24±0.11,which was significantly lower than the control group 1.00±0.07 and the negative control group 0.98±0.12(P<0.001);The 72 h OD value of the experimental group was 0.72±0.06,which was significantly lower than that of the control group 1.20±0.10 and the negative control group 1.27±0.08(P<0.001).The number of colony cells formed in the experimental group was 16.33±2.52 significantly lower than that of the control group 44.67±4.51 and the negative control group 47.12±4.05(P<0.001);The number of invaded cells in the experimental group was 54.33±9.50,which was lower than 102.14±12.50 in the control group and 105.62±14.35 in the negative control group(P<0.001);Compared with the control group and the negative control group,the expression level of E-cadherin protein in the experimental group increased,the expression level of Vimentin and N-cadherin protein decreased.Conclusion:The expression level of Twist in liver cancer is increased,knocking down its expression level can inhibit cell proliferation,invasion and epithelial-mesenchymal transition.