shRNA沉默GnT-Ⅴ基因表达对前列腺癌PC-3细胞增殖和凋亡的影响

Effect of silencing GnT-Ⅴ gene expression by shRNA on proliferation and apoptosis of prostate cancer cell line PC-3

目的构建针对N-乙酰氨基葡萄糖转移酶Ⅴ(GnT-Ⅴ)的小片段发夹状RNA(shRNA)表达质粒,研究shRNA表达质粒沉默GnT-Ⅴ基因后对前列腺癌PC-3细胞增殖和凋亡的影响。方法设计针对GnT-Ⅴ基因的小干扰RNA(siRNA)靶序列,构建shRNA表达载体并转染PC-3细胞,通过G418筛选,建立稳定表达GnT-Ⅴ基因的细胞株,采用RT-PCR和蛋白质印迹检测GnT-ⅤmRNA和蛋白的表达,并通过CCK-8增殖实验、流式细胞仪评价GnT-ⅤshRNA对前列腺癌PC-3细胞增殖和凋亡的影响。结果成功构建了GnT-ⅤshRNA表达质粒,且该质粒明显下调GnT-Ⅴ的表达;PC-3细胞GnT-Ⅴ/1079的mRNA和蛋白质水平的抑制率分别为76.5%和67.0%,对PC-3细胞呈明显抑制效应;CCK-8增殖实验显示,与对照组相比,PC-3GnT-Ⅴ/1079的增殖受到明显抑制(P<0.01),以48h为著;流式细胞仪检测结果表明,PC-3GnT-Ⅴ/1079的凋亡率明显增加(P<0.05)。结论 shRNA GnT-Ⅴ能显著降低GnT-Ⅴ基因的表达水平,从而有效抑制PC-3细胞增殖,并促进细胞凋亡,该GnT-Ⅴ的siRNA序列可能成为治疗前列腺癌的有效靶点。

Objective To construct expression vectors of small hairpin RNA （shRNA） aimed at N acetylglucosaminyltransferase V （GnT-V） gene, and to investigate effects of GnT-V shRNA on proliferation and apoptosis of prostate cancer PC-3 cells. Methods To design siRNA according to the coding sequence of GnT-V gene, shRNA expressionvectors were constructed and transfected into PC-3 cells, cell lines which stably expressing low level of GnT-V were established by G418 screening. RT-PCR and Western blot were applied respectively to detect the GnT-V mRNA and protein expression, cell proliferation and apoptosis of prostate cell line were examined by CCK-8 assay and flow cy- tometry respectively. Results GnT-V shRNA expression piasmid was constructed successfully and pGPU6/GFP/Neo GnT-V shRNA down-regulated expression of GnT-V dramatically in PC-3 cell. The level of mRNA and protein expression of GnT-V/1079 decreased by 76.5% and 67. 0% respectively, it means depress the PC-3 cells obviously. CCK-8 assay showed proliferation of PC-3 GnT-V/1079 was suppressed obviously, compared to control group （P 〈 0. 01）, especially in 48 hours. The result of flow cytometry showed that the apoptotic rate of PC-3 GnT-V/1079 was significantly increased （P〈0.05）. Conclusions The shRNA aimed at GnT-V gene could reduce the expression of GnT-V both in the level of mRNA and protein. By this way, it can inhibit the pro liferation and promote the apoptosis of prostate cancer cell line PC-3, so the optimal GnT-V siRNA sequence segment may be provides a valid target for treating prostate cancer.