hTERT启动子的克隆及其在端粒酶阳性肺癌细胞中的靶向转录活性研究  

Study on Cloning of hTERT Promoter and Its Targeting Transcriptional Activities in Telomerase-positive Lung Cancer Cells

作　　者：王艳萍[1] 唐小军 陈晓禾[1] 车国卫[1] 朱大兴[1] 孙芝琳[1] 周清华[1] 

机构地区：[1]四川大学华西医院肺癌分子生物实验室,成都610041

出　　处：《四川大学学报（医学版）》2006年第4期497-501,共5页Journal of Sichuan University(Medical Sciences)

基　　金：国家自然科学基金(批准号30270589;30470762)资助

摘　　要：目的克隆人端粒酶催化亚单位(hTERT)启动子序列片段,并探讨hTERT启动子在肿瘤细胞中的靶向转录活性。方法用PCR方法克隆293细胞DNA hTERT 5′端上游旁侧序列长约1.1 kb的启动子片段,经测序无误后将其克隆入荧光素酶报告质粒pGL 3-B as ic的荧光素酶基因上游,构建pGL 3-hTERT p重组质粒,瞬时转染肺癌细胞A 549、SPC-A-1、LTEPα-2、NC I-H 446、YTM LC-9、GLC-82、95D、A 2以及正常成纤维细胞M RC-5,检测荧光素酶活性。并用RT-PCR和TRAP-EL ISA方法分析各细胞hTERT mRNA的表达及其端粒酶活性。结果琼脂糖电泳显示PCR克隆的hTERT启动子片段长约1.1 kb,DNA测序结果与G enB ank中hTERT启动子DNA序列完全一致,位于hTERT基因转录起始位点上游1126 bp(-1126^-40),片段长度为1084 bp;采用双酶切和PCR两种方法鉴定pGL 3-hTERT p重组质粒,均显示构建成功;hTERT mRNA和端粒酶活性在肺癌细胞中呈不同水平的表达,而正常细胞则均为阴性;瞬时转染及荧光素酶活性检测实验显示,hTERT启动子片段在所有检测的肺癌细胞中均有高低不同的转录活性,在正常细胞M RC-5无转录活性。结论克隆的1084 bp大小的hTERT启动子片段在hTERTmRNA和端粒酶阳性的肺癌细胞中有特异性的转录活性,hTERT启动子可能作为靶向性基因治疗的调控元件。Objective To clone sequence of hTERT promoter and study its transcriptional activity and its relationship with hTERT mRNA expression and telomerase activity in various kinds of human lung cancer cells and normal cells, and to investigate the targeting transcriptional activity of hTERT promoter in tumor cells. Methods About 1.1 kb promoter of the 5＇flanking sequence of the hTERT was amplified from genomic DNA isolated from 293 cells by polymerase chain reaction （PCR）. After being confirmed by DNA sequencing, the hTERT promoter was inserted into luciferase reporter vectors （pGL3-basic） to reconstruct a recombinant named pGL3-hTERTp. Then pGL3-hTERTp was transiently transfected into lung cancer cell A549, SPC-A-1, LTEPa-2, NCI-H446, YTMLC-9, GLC-82, 95D, A2, and normal cell of MRC-5. The transcriptional activities of hTERT promoter in various cells were determined by measuring the luciferase activities, hTERT mRNA expression and telomerase activity were determined by RT-PCR and TRAP ELISA. Results Eelectrophoresis demonstrated that the hTERT promoter amplified by PCR was about 1.1 kb long, and DNA sequencing showed a sequence the same as the hTERT promoter registered in GenBank being 1084 bp in length. The recombinant of plasmid pGL3-hTERTp was confirmed by double digestion and PCR methods with correct results, hTERT mRNA and telomerase activity were expressed in all of eight lung cancer cell lines at varied levels, but not expressed in normal cell. Transient transfection assay and Luciferase assay also revealed that hTERT promoter had different transcriptional activities in various lung cancer cells, but no transcriptional activity was shown in normal cells. Conclusion 1084 bp hTERT promoter cloned has specific transcriptional activities in various telomerase-positive lung cancer cells, and it may act as control element in tumor - targeting gene therapy.

关 键 词：人端粒酶催化亚单位 启动子 靶向转录 肺癌 

分 类 号：Q785[生物学—分子生物学] R734.2[医药卫生—肿瘤]