转录因子E2F启动子的克隆及其肿瘤靶向特异性的鉴定

Preparation of transcriptional factor E2F promoter and the identification of its cancer-specificity

目的:克隆转录因子E2F的启动子,研究其针对pRb/E2F/p16调控环路缺陷肿瘤的特异性。方法:提取HepG-Ⅱ细胞基因组DNA,PCR扩增其转录因子E2F启动子(E2Fp),插入pGL3-Basic多克隆位点,构建成pGL3-E2Fp;采用Luciferase报告基因检测方法,在肿瘤细胞和正常细胞内测定E2Fp介导的Luciferase表达,以推测E2Fp肿瘤的特异性。结果:E2Fp在正常细胞内几乎没有活性,而在肿瘤细胞内具有较高的活性,两组间Luciferase报告基因表达的差异有显著性(P=0.0004)。结论:靶向pRb/E2F/p16环路缺陷肿瘤的E2Fp,具有对基因表达调控的特异性,能够提高Luciferase报告基因在肿瘤细胞中表达的靶向性。E2Fp的克隆为改进肿瘤基因治疗的疗效提供了一种可靠的工具。

Objectlve：To clone the promoter of transcriptional factor E2F （E2Fp）, and to investigate its specific activity targeting the cancers with a defective pRb/E2F/p16 regulatory pathway. Methods：E2Fp was amplified from the genome DNA of HepG-Ⅱ cells, then inserted into the multiple cloning sites of the plasmid pGL3-Basic to generate the pGL3-E2Fp. Dual-luciferase reporter assay was used to detect the expression of luciferase in cancer and normal cells, by which the E2Fp cancer-specificity was analyzed. Results： E2Fp almost had no activity in normal cells, but had strong activity in cancer cells. There was an obvious difference between the expression of luciferase in cancer and normal cells （ P = 0. 000 4 ）. Conclusion： The E2Fp targeting the cancers with a defective pRb/E2F/p16 regulatory pathway has a cancer-specificity for regulating gene expression. It can increase the specificity of luciferase gene expression in cancer cells. The cloning of E2Fp provides us a useful tool for target gene therapy of cancers.