Guoxin Yan, Bing Fan, Ronghai Zou, Jian Zhang, Xiaofeng Sun, Lei Tong, Qimin Wang, Jinhong Han, Xufei Lu, Ying Wang, Yuan Zhou, Zongxuan He, Yixiang Liao, Ning Li, Lei Cao, Zhenggang Chen
Dept. of Stomatology, Wuxi No 2. People's Hospital, Wuxi 214002, China.
Center of Stomatology, Qingdao Municipal Hospital Affiliated to Qingdao University Medical College, Qingdao 266071, China.
Hua Xi Kou Qiang Yi Xue Za Zhi. 2016 Dec 1;34(6):620-625. doi: 10.7518/hxkq.2016.06.014.
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.
SSC-4 tongue cancer cells were cultured in vitro and then transfected with small interfering RNA to knock down RhoA expression. The tested cells were divided into three groups: experimental group (experimental 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 tetrazolium assessment.
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.
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.
本研究通过RNA干扰研究RhoA基因沉默对舌癌细胞增殖和生长的影响，并探讨其可能机制。
体外培养SSC-4舌癌细胞，然后用小干扰RNA转染以敲低RhoA表达。将受试细胞分为三组：实验组（实验组1：转染RhoA-siRNA-1；实验组2：转染RhoA-siRNA-2）、阴性对照组（转染随机序列NC-siRNA）和空白对照组（转染Lipofectamine）。分别采用定量实时聚合酶链反应和蛋白质印迹法检测RhoA mRNA的表达水平。此外，通过蛋白质印迹法评估细胞周期蛋白D1、p21和p27以及RhoA蛋白的表达水平。通过评估倍增时间和噻唑蓝比色法分析增殖和生长潜能。
与阴性对照组和空白对照组相比，实验组转染siRNA后RhoA基因和蛋白的表达水平显著降低。细胞周期蛋白D1的表达显著降低，p21和p27的表达显著增加。倍增时间延长，生长潜能降低。
结果表明，RhoA基因沉默可抑制舌癌细胞的增殖，而RhoA通过调节细胞周期途径影响细胞增殖。因此，RhoA是舌癌基因治疗的潜在靶点。
