小鼠TERT启动子的克隆及在肿瘤细胞中的转录活性研究

Cloning of mTERT promoter and its transcriptional activity in tumor cells

目的:克隆不同长度的小鼠端粒酶逆转录酶(mouse telomerase reverse transcriptase,mTERT)启动子,研究其在小鼠肝癌细胞Hepa1-6、结肠癌细胞CT26、恶性黑色素瘤细胞B16和小鼠成纤维细胞NIH3T3中的转录活性。方法:以Hepa1-6细胞的基因组DNA为模板,采用PCR方法扩增不同长度的mTERT启动子片段,分别命名为mTERT1至7;然后分别克隆入荧光素酶报告基因质粒pGL3-Basic构建真核表达载体;采用脂质体Lipofectamine2000将重组质粒分别和pRL-CMV共转染Hepa1-6、CT26、B16和NIH3T3细胞,采用双荧光素酶法检测不同长度的mTERT启动子片断在细胞中的转录活性。结果:双酶切和测序鉴定均显示不同长度的mTERT启动子克隆成功及其表达载体构建成功;荧光素酶活性检测显示mTERT1至5在3种肿瘤细胞中有较高的转录活性,而在NIH3T3细胞中的活性较低,mTERT6和mTERT7在所有细胞中均很低;同一片断在不同肿瘤细胞株中活性不同。结论:mTERT核心启动子区位于ATG上游333bp内,且具有肿瘤特异性。

Objective：To clone different mouse TERT promoter segments and study its transcriptional activities in mouse hepatoma cell Hepal -6, colon cancer cell CT26, malignant melanoma cell B16 and fibroblast NIH3T3. Methods： Genomie DNA of Hepal -6 cells was used as the template,mouse telomerase reverse transcriptase （mTERT） promoter segments with different lengths were amplifed by PCR and named mTERT1 to 7 and then respectively cloned into pGL3 - Basic vectors with luciferase reporter gene to construct eukaryotic expression vectors. The recombinant vectors respectively with pRL - CMV vector were co - transfected into Hepal - 6, CT26,B16 and NIH3T3 cells. The transcription activities were analyzed by double lueiferase method. Results： The different length mTERT promoter fragments were successfully cloned and its expression vectors were succesfully constrcuted by restriction enzyme digestion and sequencing. The activities of mTERT1 to 5 were higher in three tumor cells ,but were lower in NIH3T3 cells. The activities of mTERT6 and mTERT7 were very low in all cells. The same mTERT promoter fragment had different activities in different tumor cells. Conclusion：The core region of mTERT is located in upstream 333bp of ATG and is tumor - spesific.