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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 effect of heat-induced hTERT transcriptional activity and targeting by 8-fold repeated heat shock element (8HSE). METHODS: h TERTp was cloned from human colon cancer cell by PCR. H TERTp purified by h TERTp or 8HSE was recombined with luciferase reporter vector p GL4.2 and ligated with plasmid pGL4.74 TK promoter) were co-transfected hTERT high expression of SW480 cell line and h TERT low expression of MKN28 cell line, dual luciferase assay h TERTp at 37 ℃ and 43 ℃ transcriptional activity. Results: PCR, restriction enzyme digestion and DNA sequencing showed that h TERTp clone, 8HSE synthesis and vector construction were successful. At 37 ℃, the transcriptional activity of h TERTp in SW480 cells was significantly higher than that in MKN28 cells (P <0.05), while 8HSE did not enhance the transcription activity of h TERTp, and 8HSE had an inhibitory effect on h TERT transcription in SW480 cells. At 43 ℃, the transcription activity of h TERTp was not significantly changed in both cells (P> 0.05). However, compared with h TERTp alone, the transcription activity of h TERTp modified by 8HSE was significantly increased in SW480 cells (P <0.05) But no significant change in MKN28 cells (P> 0.05). CONCLUSION: Thermal induction has no obvious effect on the transcription activity of h TERTp, but enhances the transcription activity of h TERTp modified by 8HSE. The regulatory element is targeted to tumor cells with high expression of h TERTp.