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 |
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引用本文: | 王炳卫,杨国胜,范立新,邱晓拂.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[J].中国医学文摘(检验与临床),2013(3):151-154. |
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作者姓名: | 王炳卫 杨国胜 范立新 邱晓拂 |
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作者单位: | 广东省第二人民医院泌尿外科,广州510317 |
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基金项目: | 广东省科技计划项目(20118031800108) |
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摘 要: | 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基因的小于扰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tuRNA和蛋白质水平的抑制率分别为76.5%和67.0%,对PC3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shRNAGnT-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。
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关 键 词: | 前列腺肿瘤 RNA干扰 GnT—V基因 细胞增殖 细胞凋亡 |
Effect of silencing GnT- V gene expression by shRNA on proliferation and apoptosis of prostate cancer cell line PC-3 |
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Authors: | WANG Bing -weei YANG Guo sheng FAN Li xin QIU Xiao-fu |
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Institution: | . (Department of Urology, the Second People's Hospital of Guangdong Provincial, Guangzhou 51031 7, China) |
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Abstract: | 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. |
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Keywords: | Prostatic neoplasms RNA interference GnT-V gene Cell proliferation Apoptosis |
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