基础研究(Basic Research)

热诱导对8HSE 修饰的hTERT 启动子转录活性的增强作用

Published at: 2015年第24卷第5期

王孝珑 1 , 贺赛 1 , 孙学军 1 , 周培华 1 , 郑见宝 1 , 魏光兵 1 , 崔飞博 1 , 肖宁 1 , 张立 1 , 禄韶英 1
1 西安交通大学医学院第一附属医院 普通外科,陕西 西安 710061
通讯作者 学军 孙 Email: sunxy@mail.xjtu.edu.cn
DOI: 10.3978/j.issn.1005-6947.10.3978/j.issn.1005-6947.2015.05.020
基金:
国家自然科学基金资助项目 81101874;81172362;81172359
陕西省科技统筹创新工程计划资助项目 2013KTCQ03-08

摘要

目的:探讨热诱导对8 倍重复串联热休克元件(8HSE)修饰的人端粒酶逆转录酶启动子(hTERTp)转录活性及靶向性的影响。方法:用PCR 法从人结肠癌基因组中克隆hTERTp;将单纯的hTERTp 或8HSE 修饰的hTERTp 重组于双荧光素酶报告载体pGL4.2 后,分别与内参质粒pGL4.74(含TK 启动子)共转染hTERT 高表达的SW480 细胞株和hTERT 低表达的MKN28 细胞株,双荧光素酶法检测hTERTp 在37 ℃及43 ℃下的转录活性。结果:PCR、酶切和DNA 测序表明hTERTp 克隆、8HSE 合成及载体构建成功。37 ℃ 下, 单纯hTERTp 在SW480 细胞中的转录活性明显高于MKN28 细胞(P<0.05),8HSE 对hTERTp 转录活性无增强作用,且在SW480 细胞中8HSE 对hTERT 转录活性具有抑制作用;43 ℃下,单纯hTERTp 的转录活性在两种细胞中无明显改变(P>0.05),但与单纯hTERTp 比较,8HSE 修饰的hTERTp 转录活性在SW480 细胞中明显增强(P<0.05),而在MKN28 细胞中无明显改变(P>0.05)。结论:热诱导对hTERTp 转录活性无明显影响,但对8HSE 修饰的hTERTp 转录活性有明显的增强作用,且该调控元件靶向于hTERT 高表达的肿瘤细胞。


Enhancing effect of thermal induction on transcriptional activity of 8HSE modified hTERT promoter

Abstract

Objective: To investigate the effects of thermal induction on transcriptional activity and targeting specificity of human telomerase reverse transcriptase promoter (hTERTp) modified with eight tandem repeats of consensus heat shock element (8HSE). Methods: The hTERTp was cloned from the genome of colon cancer cells by PCR amplification, and the hTERTp or hTERTp modified with 8HSE fragments were recombined into the dual luciferase reporter vector pGL4.2, which were then co-transfected with the internal control plasmid pGL4.74 (containing TK promoter) into the high hTERT expressing SW480 cells and low hTERT expressing MKN28 cells, respectively. The transcriptional activity of hTERTp in the two types of cells at the temperature of 37 ℃ and 43 ℃ were analyzed by dual-luciferase reporter assay system. Results: As identified by PCR, enzyme digestion and sequencing, the hTERTp cloning, 8HSE synthesis and plasmid vector constructions were all successful. Under the condition of 37 ℃, the transcriptional activity of the unmodified hTERTp in SW480 cells was significantly higher than that in MKN28 cells (P<0.05), while 8HSE modification exerted no enhancing effect on transcriptional activity of hTERTp, and it even inhibited the transcriptional activity of hTERTp in SW480 cells; under the condition of 43 ℃, the transcriptional activity of unmodified hTERTp in either type of cells showed no significant change (P>0.05), while compared with unmodified hTERTp, the transcriptional activity of 8HSE modified hTERTp in SW480 cells was significantly increased (P<0.05), but showed no significant change in MKN28 cells (P>0.05). Conclusion: Thermal induction has no obvious effect on transcriptional activity of hTERTp, but can significantly enhance the transcriptional activity of 8HSE modified hTERTp, which also specifically targets tumor cells with high hTERT expression.


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引用

引用本文: 孝珑 王, 赛 贺, 学军 孙, 培华 周, 见宝 郑, 光兵 魏, 飞博 崔, 宁 肖, 立 张, 韶英 禄. 热诱导对8HSE 修饰的hTERT 启动子转录活性的增强作用[J]. 中国普通外科杂志, 2015, 24(5): 717-722.
Cite this article as: WANG Xiaolong, HE Sai, SUN Xuejun, ZHOU Peihua, ZHEN Jianbao, WEI Guangbing, CUI Feibo, XIAO Ning, ZHANG Li, LU Shaoying . Enhancing effect of thermal induction on transcriptional activity of 8HSE modified hTERT promoter[J]. Chin J Gen Surg, 2015, 24(5): 717-722.