下调TSG101基因表达对胰腺癌细胞系PANC-1增殖的影响

TSG101 gene silencing can inhibit the proliferation potentiality of pancreatic cancer cell line PANC-1

目的:研究通过siRNA下调TSG101的表达对胰腺癌细胞系PANC-1生长、增殖及细胞周期的影响。方法:将合成的TSG101 siRNA片段插入至带有绿色荧光蛋白基因(GFP)的pGenesil-1质粒中,用lipo-fectamine 2000将质粒转入胰腺癌细胞系PANC-1后,Western blot鉴定siRNA对TSG101蛋白干扰效率。然后用MTT、流式细胞术检测TSG101下调的细胞生长、增殖能力及细胞周期的变化。结果:TSG101 siRNA有效下调其在胰腺癌细胞系PANC-1中的表达,下调TSG101基因的表达后显著抑制了PANC-1细胞的生长、增殖能力。与随机对照siRNA及阴性对照细胞相比,MTT分析显示有效转染TSG101 siRNA的细胞从第4天开始增殖能力明显下降(P<0.05),流式细胞术分析示细胞周期阻滞在G1期,增殖指数(PI)下降(P<0.05)。结论:下调TSG101在胰腺癌细胞系PANC-1表达后能够导致细胞周期的阻滞并有效抑制细胞生长、增殖。利用RNA干涉技术下调内源性TSG101表达有望成为一种有效的胰腺癌基因治疗方法。

Objective： To study the effects of TSG101 gene silencing on the proliferation potentiality of human pancreatic cancer cell line PANC-1.Methods： TSG101-siRNA plasmids were synthesized and cloned into the expression vector pGenesil-1 with GFP gene.Then plasmids were stably transfected into human pancreatic cancer cell line PANC-1 using lipofectamine 2000.Western blot was performed to confirm the inhibitory effect of siRNA on TSG101 protein expression.The proliferation and cell cycle of siRNA transfected PANC-1 cells and controls were evaluated by MTT and FCM respectively.Results： siRNA can obviously down-regulate the TSG101 protein expression in PANC-1 cells.And the TSG101 gene silencing can significantly inhibite the proliferation of PANC-1 cells after 4-day-culture.The results of flow cytometry showed that the proliferation index（PI） of cells transfected with TSG101-siRNA were obviously lesser than controls,which showed more in G0/G1 phase and lesser in S phase（P0.05）.Conclusion：TSG101-siRNA plasmids can effectively down-regulate TSG101 expression in PANC-1 cells,which can inhibit the potentiality of cell growth and proliferation,and may provide a novel approach for gene therapy on pancreatic cancer.