摘要
目的:通过基因克隆技术构建人前列腺癌PC3细胞系PTI1(PC3)基因[prostatecarcinomatumor inducinggene1(PC3)]的特异性短发卡shRNA(short hairpinRNA)真核表达载体,采用RNA干扰(RNAinterference,RNAi)技术,体外观察对PC3细胞系PTI1(PC3)基因的沉默作用以及干扰后对PC3细胞的体外效应.方法:采用基因克隆技术,将合成的短发卡样特异性PTI1(PC3)RNA干扰寡核苷酸序列插入真核表达载体pEGFP/U6,构建PTI1(PC3)shRNA的真核表达载体,体外转染人前列腺癌PC3细胞,48h后观察细胞生物学变化;提取转染细胞总RNA及总蛋白,行RT PCR以及West ernblot观测胞内PTI1(PC3)mRNA及蛋白水平.结果:①成功构建短发卡样PTI1(PC3)shRNA真核表达载体pEGFP/U6mPs;②转染(脂质体法)PC3细胞,48h后细胞大部分死亡;③转染48h后细胞内PTI1(PC3)mRNA水平下降;④转染48h后下调胞内PTI1(PC3)蛋白水平.结论:本实验成功构建的shRNA真核表达载体通过RNA干扰,能够干扰人前列腺癌PC3细胞内PTI1(PC3)基因的表达,抑制PTI1(PC3)蛋白的表达,降低了由PTI1蛋白可能发挥的“翻译失真性”作用,促进癌细胞的死亡,进一步揭示了PTI1在前列腺癌发生、发展过程中的显性癌基因作用.由于RNA干扰的特异性从而为临床上前列腺癌的基因治疗提供了新的可能的方法.
AIM: To construct the short-hairpin RNA (shRNA) eukaryotic expression vector specific to prostate carcinoma tumor-inducing gene 1 (PC-3) [PTI-1 (PC-3)], which is overexpressed in human prostate carcinoma PC-3 cell line, and to observe the silencing effect of the established vector on PTI-1 (PC-3). METHODS: The shRNA specific to PTI-1 (PC-3) was synthesized and inserted into pEGFP/U6 by gene recombination technology to construct the expression vector, pEGFP/U6-mPs, which was then transfected into the cultured PC-3 cells. The cell total RNA was extracted to perform RT-PCR and the whole cell protein was extracted and detected by Western blot with rabbit-anti-human PTI-1 (PC-3) polyclonal antibody. RESULTS: pEGFP/U6-mPs expression vector was successfully constructed. Lipofectamine2000 mediated gene transfection of pEGFP/U6-mPs led to cell-death at 48h after transfection. The down-regulations of both the PTI-1 (PC-3) mRNA level and the PTI-1 (PC-3) protein level were also observed. CONCLUSION: The pEGFP/U6-mPs expression vector can inhibit the expression of PTI-1 (PC-3) gene in human prostate carcinoma PC-3 cell line, which may down-regulate the level of PTI-1 protein expression. 'Translational infidelity', the role of PTI-1 (PC-3) protein, is probably degraded, which may be responsible for the death of the cancer cells. The PTI-1 (PC-3) gene is further confirmed as a dominant-acting oncogene. The specificity of the RNA interference may lead to a novel approach of clinical gene therapy in treating prostate carcinoma.
出处
《第四军医大学学报》
北大核心
2005年第9期824-827,共4页
Journal of the Fourth Military Medical University
