沉默RohA基因对舌鳞状细胞癌细胞增殖的影响

摘要

目的 通过RNA干扰技术沉默RhoA基因从而探讨RhoA对舌癌细胞增殖和生长的影响及其作用机制.方法 体外培养舌鳞状细胞癌SCC-4细胞,以小分子干扰RNA转染沉默RhoA基因的表达.实验分为3组:实验组(又分为实验1组和实验2组,脂质体分别转染对应序列1的RhoA-siRNA和序列2的RhoA-siRNA)、阴性对照组(脂质体转染NC-siRNA)和空白对照组(不转染siRNA).采用实时定量聚合酶链反应技术检测SCC-4细胞转染后RhoA mRNA的表达,Western blot检测RhoA、Cyclin D1、p21和p27蛋白的表达,四唑盐比色法检测舌癌细胞生长水平和倍增时间.结果 与阴性对照组和空白对照组相比,实验组舌癌细胞的RhoA基因及蛋白表达降低,p21、p27蛋白表达升高,Cyclin D1蛋白表达降低,细胞倍增时间延长,增殖能力降低(P<0.05).结论 沉默RhoA基因可以抑制舌癌细胞的增殖和生长,RhoA基因通过调控细胞周期信号转导途径影响舌癌细胞的增殖,RhoA基因可以成为舌癌基因治疗的靶点.%Objective This study investigated the effect of RhoA silencing through RNA interference on proliferation and growth of tongue cancer cells, as well as explored the possible mechanisms of this effect.Methods SSC-4 tongue cancer cells were culturedin vitro and then transfected with small inter-fering RNA to knock down RhoA expression. The tested cells were divided into three groups: experimental group (experi-mental group 1: transfected with RhoA-siRNA-1; experi-mental group 2: transfected with RhoA-siRNA-2), negative control group (transfected by random sequence NC-siRNA), and blank control group (transfected with Lipofectamine).The expression levels of RhoA mRNA were respectively measured by quantitative real-time polymerase chain reaction and western blot assay. Moreover, the expression levels of cyclin D1, p21, and p27 and RhoA protein were evaluated by Western blot assay. Proliferation and growth potentiality were analyzed through evaluation of doubling times and methyl thiazolyl tetra-zolium assessment.Results The expression levels of RhoA gene and protein of experimental groups significantly decreased following siRNA transfection compared with those in the negative and blank control groups. The expression of cyclin D1 decreased significantly and that of p21 and p27 increased significantly. The doubling time was extended and the growth potentiality decreased.Conclusion The results indicated that RhoA silencing can inhibit proliferation of tongue cancer cells, whereas RhoA affects cell proliferation by regulating the cell cycle pathway. Thus, RhoA is a potential target in gene therapy for tongue cancer.