siRNA沉默E2F3基因对人膀胱癌细胞的增殖抑制作用

Inhibitory effect of siRNA silencing E2F3 gene on proliferation of human bladder cancer cells

目的:探讨siRNA干扰E2F3基因对膀胱癌细胞(5637细胞)增殖的抑制作用,阐明E2F3基因沉默对5637细胞增殖抑制作用的生物学机制。方法:化学合成3对编码短发夹RNA序列的、靶向E2F3基因的寡核苷酸链,经BamHⅠ、HindⅢ双酶切克隆至pRNAT-U6.1/Neo系统,重组构建E2F3siRNA的RNAi质粒,以质粒转染DH5α菌株筛选得到稳定表达siRNA的细胞,转染5637细胞E2F3基因沉默,RT-PCR及Western blotting检测E2F3表达,流式细胞术检测5637细胞周期,采用Annexin-V/PS双染法检测细胞凋亡率。结果:RT-PCR及Western blotting检测干扰后5637细胞E2F3基因表达抑制,3条干扰序列对5637细胞周期抑制且G1期细胞比例增高,与阴性转染对照组比较差异有显著性(P<0.05和P<0.001);细胞凋亡率增加,与空白对照组及阴性转染对照组比较差异有显著性(P<0.001)。结论:siRNA干扰E2F3基因对膀胱癌5637细胞具有明显增殖抑制和促进凋亡的作用。

Objective To investigate the inhibitory effect of siRNA interfering E2F3 gene on proliferation of bladder cancer 5637 cells,and clarify the biomechanism of inhibitory effect of E2F3 gene silencing on the proliferation of 5637 cells.Methods There pairs of nucleotide chains coding short RNA sequence and targeting E2F3 gene were chemically synthesized and cloned into pRNAT-U6.1/Neo digested with BamHⅠand Hind Ⅲ. RNAi plasmid（pRNAT-U6.1-E2F3/Neo） was constructed.The reconstructed RNAi plasmids were identified by electrophoresis and confirmed by sequencing analysis. The recombinant plasmids were transformed into strain DH5α,and the recombinant pRNAT-U6.1/Neo was transfected into 5637 cells. Before and after transfection,the levels of E2F3 mRNA and E2F3 protein expression were detected by RT-PCR and Western blotting,the cell cycle was assessed by flow cytometry,the apoptotic rate was detected by Annexin-V/PS kit. Results Compared with control group,the E2F3 gene expression was markedly down-regulated in 5637 cells following RNA interference treatment（ P0.001）,the percentage of cells at G1 phase and the apoptotic rate were up-regulated in transfection cells group,there were significant difference between RNAi cells groups and control group（P0.001）. Conclusion siRNA can significantly inhibit the E2F3 gene expression in bladder cancer 5637 cells,decrease cell proliferation ,arrest cell cycle and increase apoptotic rate.