RNA干扰环指蛋白2基因抑制胰腺癌细胞系PANC-1增殖和迁移

摘要

目的 研究沉默环指蛋白2(RNF2)基因对人胰腺癌细胞系PANC-1增殖、迁移、周期和凋亡的影响及可能机制.方法 用siRNA-RNF2转染PANC-1细胞沉默RNF2表达,同时设立转染无义序列(siRNA-NC)的空转染组及不进行任何处理的空白对照组(mock).荧光定量PCR检测RNF2 mRNA表达;Western blot检测RNF2和p53表达;MTS实验和划痕实验分别检测细胞增殖和迁移能力;流式细胞计量术检测细胞转染率、凋亡率和细胞周期.结果 相比正常胰腺导管上皮细胞,RNF2在胰腺癌细胞系中高表达(P<0.05).转染siRNA-RNF2的PANC-1细胞与阴性对照组比较,RNF2 mRNA及蛋白表达下调;细胞增殖被抑制(P<0.05);细胞迁移能力下降(P<0.05);细胞凋亡率增高(P<0.05);G0/G1期细胞比例上升,S期和G2/M期细胞比例则降低(P<0.05).此外,转染siRNA-RNF2显著下调PANC-1细胞中p53蛋白的表达.结论 siRNA-RNF2特异性下调胰腺癌细胞RNF2表达,显著抑制细胞增殖和迁移,结果提示RNF2可能成为胰腺癌基因治疗的一个新靶点.%Objective To investigate the effect of siRNA-mediated silencing of RNF2 on cell proliferation , migra-tion, cell cycle and apoptosis in human pancreatic cancer PANC-1 cells and its possible mechanism .Methods The siRNA interference was used to down-regulate RNF2 expression.Meanwhile, there were also empty transfection group whose cells were transfected with the control siRNA and mock group without any treatment .The result of transfection was evaluated by fluorescence microscope .The expression of RNF2 mRNA was detected by RT-qPCR. Western blot was applied to detect the expression of RNF 2 and p53.Cell proliferation and migration were analyzed by MTS assay and cell scratch assay , respectively .The transient transfection efficiency , apoptosis rate and cell cy-cle were measured by flow cytometry .Results Compared to the normalized human pancreatic duct epithelial cells , RNF2 expression in pancreatic cancer cells were higher ( P<0.05) .The expression of RNF2 mRNA and protein was decreased in PANC-1 cells by siRNA-RNF2 at 48 h post-transfection.Transfection with siRNA-RNF2 inhibited the proliferation and migration of PANC-1 cells (P<0.05), induced cell apoptosis (P<0.05), increased cell counts in phase G0/G1 and decreased in S and G2/M phase (P<0.05).What's more, after siRNA-RNF2 transfec-tion, the expression of p 53 protein was decreased .Conclusions siRNA-RNF2 can specifically knockdown the ex-pression of RNF2 gene and then inhibit the proliferation and migration of PANC-1 cells.These results indicate RNF2 may be a potential target of gene therapy for pancreatic cancer .