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目的研究干扰Ras同源物基因家族成员B(Ras homolog gene family member B,RhoB)表达后对肾透明细胞癌(renal clear cell carcinoma,RCCC)细胞生长及迁移能力的影响。方法检测RhoB在RCCC细胞中的蛋白表达水平,采用脂质体转染方法转染RhoB真核表达载体pcDNA3.0-Flag-RhoB和沉默RhoB的小干扰RNA(small interfering RNA,siRNA)即si-RhoB序列及相应空载体和对照序列,应用Western Blot法检测转染后RhoB的蛋白表达情况,通过3-(4,5-二甲基吡啶-2-基)-5-(3-羧基甲氧基苯基)-2-(4-磺苯基)-2H-四唑[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sugophenyl)-2H-tetrazolium,MTS]法、平板克隆和Transwell小室细胞迁移实验检测细胞生长和细胞迁移能力的变化。结果 RhoB在RCCC细胞中表达降低。与转染空载体组比较,转染pcDNA3.0-Flag-RhoB重组质粒组RhoB的蛋白表达水平明显上调;与转染对照组比较,si-RhoB转染组RhoB的蛋白表达水平明显下调。在RCCC细胞786-O和Caki-1中,上调RhoB的表达后,在4、5、6 d经MTS法测定光密度值明显降低(P<0.05);在786-0细胞中上调RhoB表达后细胞克隆数明显减少;转染48 h后Transwell细胞迁移数明显减少(P<0.05)。而下调RhoB表达后,细胞生长和Transwell细胞迁移数没有明显变化。在相对高表达RhoB的正常人肾小管上皮细胞中下调RhoB表达后细胞生长和细胞迁移数明显增多。结论过表达RhoB后明显抑制RCCC细胞生长和迁移能力。
Objective To investigate the effect of RhoB expression on the growth and migration of renal clear cell carcinoma (RCCC) cells after interference with RhoB gene expression. Methods The protein expression of RhoB in RCCC cells was detected by lipofectamine. The recombinant plasmid was transfected into the eukaryotic expression vector pcDNA3.0-Flag-RhoB by RhoB and the small interfering RNA (siRNA) RhoB sequence and the corresponding empty vector and control sequence, the protein expression of RhoB after transfection was detected by Western Blot. The expression of RhoB was detected by 3- (4,5-dimethylpyridin-2-yl) -5- (3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sugophenyl) 2H-tetrazolium, MTS] assay, plate clone and Transwell cell migration assay were used to detect cell growth and cell migration. As a result, RhoB expression was decreased in RCCC cells. Compared with the vector transfected group, the protein expression of RhoB in pcDNA3.0-Flag-RhoB recombinant plasmid group was significantly up-regulated; compared with the transfected control group, the protein expression of RhoB in si-RhoB transfected group was significantly down-regulated. In RCCC cells 786-O and Caki-1, after RhoB expression was up-regulated, the optical density was significantly decreased (P <0.05) by MTS method on the 4th, 5th and 6th day. After RhoB expression was up-regulated in 786-0 cells The number of cell clones decreased significantly. Transwell cell migration was significantly decreased 48 h after transfection (P <0.05). However, there was no significant change in cell growth and transwell cell migration when RhoB expression was down-regulated. RhoB expression in normal human renal tubular epithelial cells with relatively high expression of RhoB significantly increased cell growth and cell migration. Conclusion Overexpression of RhoB significantly inhibits the growth and migration of RCCC cells.