抑制TPD52基因表达对结直肠癌细胞凋亡及ROS含量的影响

Effect of inhibiting TPD52 on apoptosis and ROS content in colorectal cancer cells

目的:探讨抑制肿瘤蛋白D52(TPD52)基因表达对结直肠癌细胞活力、凋亡率及活性氧(reactive ox-ygen species,ROS)含量的影响及机制。方法:正常结肠NCM460细胞作为对照细胞,分别用RT-PCR及Western blotting检测人结直肠癌Caco2、SW480、HCT116、HT29细胞TPD52的mRNA及蛋白表达。以Lipo-fectamine^(TM)2000为载体,将抑制TPD52表达的siRNA(TPD52-siRNA)及阴性对照siRNA(NC)转染HCT116细胞,转染48 h,MTT检测细胞活力,流式细胞术检测细胞凋亡率及ROS含量; Western blotting检测各组细胞增殖相关蛋白Ki67和PCNA及凋亡相关蛋白Bax和Survivin的蛋白表达。结果:与正常结肠NCM460细胞比较,Caco2、SW480、HCT116、HT29细胞中TPD52的mRNA及蛋白表达均明显升高,差异均具有统计学意义(P<0. 05)。转染TPD52-siRNA的HCT116细胞TPD52的mRNA及蛋白表达均明显降低,与空白对照组和NC组比较差异具有统计学意义(P <0. 05)。与NC组比较,TPD52-siRNA组细胞活力明显降低,凋亡率升高,ROS含量升高,Ki67、PCNA和Survivin的蛋白表达明显降低,Bax的蛋白表达明显升高,差异均具有统计学意义(P <0. 05)。结论:TPD52基因表达抑制可降低结直肠癌细胞活力和诱导凋亡,机制可能与细胞内ROS水平提高及Ki67、PCNA、Survivin表达下调和Bax表达上调有关。

Objective: To investigate the effect of inhibition of TPD52 gene expression on cell viability,apoptosis rate and ROS content in colorectal cancer cells. Methods: Normal colon NCM460 cells were used as control cells,and RT-PCR and Western blotting were used to detect the mRNA and protein expression of TPD52 in Caco2,SW480,HCT116,HT29 cells in colorectal cancer.Lipofectamine^TM 2000 was used as a vector.siRNA inhibition of TPD52 expression(TPD52-siRNA) and negative control siRNA (NC group) were transfected into HCT116 cells.MTT was used to detect cell viability cells transfected for 48 h,and apoptosis rate and ROS content were detected by flow cytometry.Protein expression of Ki67,PCNA,Bax and Survivin were detected by Western blotting. Results: Compared with normal colon NCM460 cells,the mRNA and protein expression of TPD52 in Caco2,SW480,HCT116 and HT29 cells increased significantly,and the difference was statistically significant ( P <0.05).The mRNA and protein expression of TPD52 in HCT116 cells transfected with TPD52-siRNA were significantly decreased,and the difference was statistically significant compared with that of the blank control group and the NC group ( P <0.05).Compared with the NC group,the cell vitality decreased obviously in TPD52-siRNA group,the apoptosis rate increased,the ROS content increased,the protein expression of Ki67,PCNA and Survivin decreased obviously,and the protein expression of Bax increased obviously,the difference was statistically significant ( P <0.05). Conclusion: The inhibition of TPD52 gene expression can reduce the activity of colorectal cancer cells and induce apoptosis.The mechanism may be related to the increase of intracellular ROS level and down regulation of Ki67,PCNA and Survivin expression and up regulation of Bax expression.