Wang Yan-ping, Tang Xiao-jun, Chen Xiao-he, Che Guo-wei, Zhu Da-xing, Sun Zhi-lin, Zhou Qing-hua
Laboratory of Lung Cancer Molecular Biology, West China Hospital, Sichuan University, Chengdu 610041, China.
Sichuan Da Xue Xue Bao Yi Xue Ban. 2006 Jul;37(4):497-501.
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.
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.
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.
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.
克隆人端粒酶逆转录酶（hTERT）启动子序列，研究其转录活性以及与各种人肺癌细胞和正常细胞中hTERT mRNA表达及端粒酶活性的关系，并探讨hTERT启动子在肿瘤细胞中的靶向转录活性。
通过聚合酶链反应（PCR）从293细胞基因组DNA中扩增hTERT 5'侧翼序列约1.1 kb的启动子。经DNA测序确认后，将hTERT启动子插入荧光素酶报告载体（pGL3-basic）中构建重组体pGL3-hTERTp。然后将pGL3-hTERTp瞬时转染至肺癌细胞A549、SPC-A-1、LTEPa-2、NCI-H446、YTMLC-9、GLC-82、95D、A2以及正常细胞MRC-5。通过检测荧光素酶活性来测定hTERT启动子在各种细胞中的转录活性。采用逆转录聚合酶链反应（RT-PCR）和端粒重复序列扩增法酶联免疫吸附测定（TRAP ELISA）检测hTERT mRNA表达和端粒酶活性。
电泳显示PCR扩增的hTERT启动子约1.1 kb长，DNA测序表明其序列与GenBank中登记的长度为1084 bp的hTERT启动子序列一致。通过双酶切和PCR方法确认质粒pGL3-hTERTp重组体结果正确。hTERT mRNA和端粒酶活性在所有8种肺癌细胞系中均有不同程度表达，但在正常细胞中未表达。瞬时转染实验和荧光素酶检测还显示，hTERT启动子在各种肺癌细胞中具有不同的转录活性，但在正常细胞中无转录活性。
克隆的1084 bp hTERT启动子在各种端粒酶阳性肺癌细胞中具有特异性转录活性，可能作为肿瘤靶向基因治疗中的调控元件。
