E-box元件修饰端粒酶核心启动子序列及体外转录活性分析(英文)  

hTERT Core Promoter Modified by E-boxes Element and Transcriptional Activity Assay in vitro

作　　者：杨应斌[1,2] 蔡绍皙[1] 于淑惠[1,3] 杨力[1,4] 李军[1] 余松涛[5] 戴小珍[1] 王星星[1] 晏小清[1] 辛鑫[2] 宋国立[1,4] 

机构地区：[1]重庆大学生物工程学院,重庆400044 [2]西南大学生命科学学院,重庆400715 [3]西南大学图书馆,重庆400715 [4]加州大学骨外科与生物工程系 [5]第三军医大学西南医院消化科,重庆400038

出　　处：《中国生物化学与分子生物学报》2009年第6期510-514,共5页Chinese Journal of Biochemistry and Molecular Biology

基　　金：Supported by National Natural Science Foundation of China (No.10872224)~~

摘　　要：人类端粒酶启动子(hTERT启动子)在肿瘤基因治疗中的有效性已经得到了证实.然而,hTERT启动子有限的肿瘤靶向转录活性困扰着它的临床应用.早期研究已经揭示,核心hTERT启动子上的-34位E-box元件与该启动子的肿瘤靶向转录活性有关.为进一步探索核心hTERT启动子序列3′端富余E-box元件是否能提高启动子的肿瘤靶向转录能力,用化学合成方法在野生型hTERT(WT-hTERT)核心启动子片段(编码蛋白起始子ATG上游-268bp～-10bp)的3′端接入3个E-box序列,构建成修饰型hTERT(Mod-hTERT)启动子.然后,分别用WT-hTERT和Mod-hTERT启动子去调控增强型绿色荧光蛋白(EGFP)及荧光素酶报告基因在293FT、HepGⅡ、SGC7901、U2OS、以及原代培养人成纤维细胞(PHF)中表达.结果表明,在Mod-hTERT启动子的各实验组细胞中,能够在端粒酶阳性的293FT、HepGⅡ及SGC7901细胞组中观测到EGFP的表达,而在端粒酶阴性的U2OS及PHF细胞组中没有观测到EGFP的表达;在端粒酶阳性的293FT、HepGⅡ和SGC7901细胞株中,Mod-hTERT启动子调控下的荧光素酶活性要高于WT-hTERT启动子组(P<0.01);而在端粒酶阴性的U2OS细胞组中,Mod-hTERT启动子调控下的荧光素酶活性则低于WT-hTERT启动子组(P<0.01);在PHF细胞组中,Mod-hTERT启动子组与WT-hTERT启动子组的荧光素酶活性差异不显著(P>0.05).研究提示,在3′端增加E-box元件可以提高核心hTERT启动子序列的肿瘤靶向转录活性.The effective clinical application of human telomerase promoter （ hTERT promoter） in tumor gene therapy is limited by insufficient tumor-targeting transcription activity at - 34 E-box of its core promoter. To test whether it can be enhanced by additioanl E-boxes, a wild-type core hTERT （WT-hTERT） promoter （ - 268 to - 10 upstream of ATG start codon） and a modified hTERT （Mod-hTERT） promoter （with a triplet of E-boxes ligated to 3′ WT-hTERT） were synthesized. Their fusions with enhanced green fluorescent protein （EGFP） or luciferase were constructed and transfected into 293FT, HepG Ⅱ , SGC7901, U2OS, and the primary culture human fibroblasts （PHF）, respectively. The results showed that in the Mod-hTERT-driven cells, EGFP expression could be observed in hTERT-positive cells like 293FT, HepG Ⅱ , and SGC7901 cells, but not in hTERT-negative cells as U2OS and PHF. The luciferase activity in Mod-hTERT transfected cells was significantly higher than that of the WT-hTERT in the hTERT positive cell lines of 293FT, HepG Ⅱ , and SGC7901（P 〈 0.01）, but lower in the hTERT-negative cell lines of U2OS （P 〈 0.01）. In PHF, no significant difference was found （ P 〉 0.05 ）. Our study indicates that multiple E-boxes improve the tumor-targeting transcription activity of the core hTERT promoter.

关 键 词：人类端粒酶启动子 E-BOX 荧光素酶 增强型绿色荧光蛋白 肿瘤靶向性 

分 类 号：Q78[生物学—分子生物学] Q81