热诱导对8HSE修饰的hTERT启动子转录活性的增强作用

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

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(h TERTp)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h TERTp;将单纯的h TERTp或8HSE修饰的h TERTp重组于双荧光素酶报告载体p GL4.2后,分别与内参质粒p GL4.74(含TK启动子)共转染h TERT高表达的SW480细胞株和h TERT低表达的MKN28细胞株,双荧光素酶法检测h TERTp在37℃及43℃下的转录活性。结果:PCR、酶切和DNA测序表明h TERTp克隆、8HSE合成及载体构建成功。37℃下,单纯h TERTp在SW480细胞中的转录活性明显高于MKN28细胞(P<0.05),8HSE对h TERTp转录活性无增强作用,且在SW480细胞中8HSE对h TERT转录活性具有抑制作用;43℃下,单纯h TERTp的转录活性在两种细胞中无明显改变(P>0.05),但与单纯h TERTp比较,8HSE修饰的h TERTp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h TERTp转录活性无明显影响,但对8HSE修饰的h TERTp转录活性有明显的增强作用,且该调控元件靶向于h TERT高表达的肿瘤细胞。

Objective：To investigate the effects of thermal induction on transcriptional activity and targeting specificity of human telomerase reverse transcriptase promoter（h TERTp） modified with eight tandem repeats of consensus heat shock element（8HSE）.Methods：The hT ERTp was cloned from the genome of colon cancer cells by PCR amplification,and the hT ERTp or hT ERTp modiifed with 8HSE fragments were recombined into the dual luciferase reporter vector pG L4.2,which were then co-transfected with the internal control plasmid pG L4.74（containing TK promoter） into the high hT ERT expressing SW480 cells and low hT ERT expressing MKN28 cells,respectively.The transcriptional activity of hT ERTp in the two types of cells at the temperature of 37 ℃ and 43 ℃ were analyzed by dual-luciferase reporter assay system.Results：As identiifed by PCR,enzyme digestion and sequencing,the hT ERTp cloning,8HSE synthesis and plasmid vector constructions were all successful.Under the condition of 37 ℃,the transcriptional activity of the unmodiifed hT ERTp in SW480 cells was signiifcantly higher than that in MKN28 cells（P0.05）,while 8HSE modification exerted no enhancing effect on transcriptional activity of hT ERTp,and it even inhibited the transcriptional activity of hT ERTp in SW480 cells; under the condition of 43 ℃,the transcriptional activity of unmodiifed hT ERTp in either type of cells showed no signiifcant change（P0.05）,while compared with unmodiifed hT ERTp,the transcriptional activity of 8HSE modiifed hT ERTp in SW480 cells was signiifcantly increased（P0.05）,but showed no signiifcant change in MKN28 cells（P0.05）.Conclusion：Thermal induction has no obvious effect on transcriptional activity of hT ERTp,but can significantly enhance the transcriptional activity of 8HSE modified hT ERTp,which also specifically targets tumor cells with high hT ERT expression.