RNA干扰沉默Spl基因抑制前列腺癌细胞中CD59的表达

The effects of RNA interference-induced Spl gene silence on expression of CD59 in prostate cancer cell

目的采用RNA干扰技术沉默前列腺癌PC-3细胞转录因子Sp1的表达,观察其对CD59的表达影响。方法构建靶向人Spl基因的干扰载体(pSUPER-siSp1),脂质体介导转染PC-3细胞,荧光倒置显微镜下观察转染效率,RT-PCR及Western-blotting检测其对Sp1及CD59蛋白质水平的影响,流式细胞仪检测CD59的表达,MTT法检测CD59抗补体活性的变化。结果成功构建针对Spl的干扰载体,转染后能够有效抑制前列腺癌PC-3细胞中Sp1与CD59蛋白的表达,与对照组相比,差异有统计学意义(P<0.05)。MTT实验显示,下调Spl表达可显著抑制CD59抗补体活性。结论 Spl基因沉默能够抑制人前列腺癌CD59的表达,为前列腺癌基因治疗提供新的思路和手段。

To analyze the role of Sp1 in the expression of CD59,we constructed recombinant vectors expressing siRNA that target Sp1 gene and a stable-inhibit cell line PC-3.The constructed recombinant plasmid of pSUPER-siSp1 was transfected into PC-3 cells by Lipofectamine,while the transfection efficiency was observed under fluorescence confocal microscopy.The expression levels of Spl and CD59 protein on the PC-3 cells were examined by RT-PCR and Western blotting respectively.Furthermore,the expression level of CD59 protein was examined by flow cytometry.The proliferation ability of PC-3 cell line was evaluated by MTT assay.We found the recombinant vectors could significantly reduce the expressions of Sp1 and CD59 protein in PC-3,as compared with the control group（P 0.05）.MTT showed that CD59＇s protection to complement-mediated cytolysis decreased.All the results indicated that silencing Sp1 gene by the RNAi technology can actively inhibit the expression of CD59 in PC-3 cell.The successful application of Sp1 siRNA extends the list of available therapeutic modalities in the treatment of human prostate cancer.