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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 m RNA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 To investigate the effect of RhoA on the proliferation and growth of tongue cancer cells by silencing the RhoA gene by RNAi technique and its mechanism. Methods Squamous cell carcinoma SCC-4 cells were cultured in vitro and the expression of RhoA gene was silenced by small interfering RNA. The experiment was divided into three groups: experimental group (divided into experimental group 1 and experimental group 2, liposome transfection corresponding sequence 1 RhoA-siRNA and sequence 2 RhoA-siRNA respectively), negative control group NC-siRNA) and blank control group (not transfected with siRNA). The expression of RhoA m RNA in SCC-4 cells was detected by real-time quantitative polymerase chain reaction. The expression of RhoA, Cyclin D1, p21 and p27 proteins was detected by Western blot. The growth of tongue cancer cells was detected by tetrazolium salt colorimetric assay. Double the time. Results Compared with the negative control group and the blank control group, the expression of RhoA gene and protein, the expression of p21 and p27 protein, the expression of Cyclin D1, the cell doubling time and the proliferative ability of tongue cancer cells in experimental group were decreased (P < 0.05). Conclusion Silencing RhoA gene can inhibit the proliferation and growth of tongue cancer cells. RhoA gene can influence the proliferation of tongue cancer cells by regulating cell cycle signal transduction pathway. RhoA gene may be the target of tongue cancer gene therapy.