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目的应用RNA干扰技术,体外沉默二牛龙牛儿基转移酶Ⅰ(GGTase-Ⅰ),研究其在舌癌迁移、侵袭中的作用。方法登录Genebank确定人GGTase-Ⅰ基因序列,针对GGTase-Ⅰ的基因序列设计并构建3条si RNA,分别将RNA干扰组(GGTase-Ⅰsi RNA1、GGTase-Ⅰsi RNA2、GGTase-Ⅰsi RNA3)、阴性对照组(NC-si RNA)和空白对照组转染至舌癌细胞Cal-27,实时定量聚合酶链反应(q RT-PCR)和蛋白质印迹法(Western blot)检测各组细胞转染后GGTase-Ⅰ、Rho A基因的m RNA、蛋白表达;选取沉默效率最高的一组si RNA作为实验组,蛋白质印迹法检测各组细胞基质金属蛋白酶(MMP)-2、MMP-9的表达,GST-pull down实验检测GTP-Rho A蛋白的表达,划痕实验检测细胞迁移能力变化,Transwell小室检测细胞侵袭能力变化。结果干扰后细胞的GGTase-Ⅰm RNA、蛋白表达明显下降(P<0.05),Rho A m RNA和蛋白表达无明显改变,MMP-2、MMP-9、GTP-Rho A的表达下降,迁移、侵袭能力明显下降(P<0.05)。结论抑制GGTase-Ⅰ表达,可降低舌癌细胞的迁移、侵袭能力,对GGTase-Ⅰ的深入研究可能为舌癌的治疗提供新的有效分子靶点。
Objective To investigate the role of GGTase-Ⅰ in the silencing of the migration and invasion of tongue cancer by RNA interference. Methods The sequence of human GGTase-Ⅰ gene was determined by logging on Genebank. Three si RNAs were designed and constructed according to the GGTase-Ⅰ gene sequence. The RNA interference group (GGTase-ⅠsiRNA1, GGTase-ⅠsiRNA2, GGTase-ⅠsiRNA3) (NC-si RNA) and blank control group were transfected into tongue cancer cells Cal-27, real-time quantitative polymerase chain reaction (q RT-PCR) and Western blot were used to detect the expression of GGTase- Ⅰ, Rho A gene m RNA and protein expression; selected a group of the most efficient silencing si RNA as the experimental group, Western blotting assay the expression of matrix metalloproteinase (MMP -2, MMP-9, GST-pull down assay to detect the expression of GTP-Rho A protein. Scratch assay was used to detect the change of cell migration ability. Transwell chamber was used to detect the change of cell invasiveness. Results The mRNA and protein expression of GGTase-ⅠmRNA in the cells were significantly decreased (P <0.05), while the expression of Rho A m RNA and protein did not change significantly. The expressions of MMP-2, MMP-9 and GTP-Rho A were decreased, Ability decreased significantly (P <0.05). Conclusion Inhibition of GGTase-Ⅰ expression can reduce the migration and invasion ability of tongue cancer cells. Further study on GGTase-Ⅰ may provide a novel and effective molecular target for the treatment of tongue cancer.