不同修饰型hTERT启动子在多种肿瘤细胞中的转录活性研究

Study on the transcriptional activities of modified hTERT promoters in tumor cells

目的:对人端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT)启动子进行不同修饰,然后对其在不同肿瘤细胞和原代培养的人牙龈成纤维细胞(human gingival fibroblasts,HGF)中的转录活性进行比较。方法:常规分子生物学方法克隆hTERT启动子区-378至+77之间序列和CWV增强子,采用SV40增强子、CWV增强子、SV40-CWV双增强子、Myc-Max反应元件(Myc-Max response elements,MMRE)、MMRE和SV40增强子联合对hTERT启动子分别进行修饰,分别构建其荧光素酶报告基因表达载体;采用脂质体Li-pofectamine2000将重组质粒分别和pRL-CMV共转染不同肿瘤细胞和原代培养的牙龈成纤维细胞,双荧光素酶法检测hTERT启动子的转录活性。结果:酶切和测序鉴定hTERT启动子、CWV增强子克隆成功,以及荧光素酶表达载体构建成功;荧光素酶活性检测显示:野生型hTERT启动子不同肿瘤细胞中相对转录活性从9.9%到24.4%有所不同,经不同修饰后活性大多有所提高,分别是野生型hTERT启动子的倍数:SV40增强子1.3至4.9倍;CWV增强子4.0至42.8倍;CWV和SV40联合增强子2.0至49.2倍;单一MMRE0至5.5倍,MMRE和SV40增强子联合为1.2至8.4倍。野生型和修饰后的hTERT启动子在原代培养的牙龈成纤维细胞转录活性均较低。同一hTERT启动子在不同肿瘤细胞中转录活性不同。结论:不同修饰的hTERT启动子相对野生型hTERT转录活性大多有所提高,其中CMV增强子或SV40-CMV双增强子修饰的hTERT启动子提高幅度最大,用于靶向性肿瘤基因治疗具有巨大潜力。

Objective：To study the transcriptional activities of modified hTERT promoters in different tumor cell lines and primary human gingival fibroblasts.Methods：The sequence between-378bp to ＋77bp of hTERT promoter region and CMV enhancer were cloned using molecular biological methods.The hTERT promoter was modified with SV40 enhacer,CWV enhancer,SV40-CWV double enhancer,Myc-Max response elements（MMRE）,and MMRE combined with SV40 enhacer and then these modified hTERT promoters were constructed into eukaryotic luciferase expression vectors.The recombinant vectors and pRL-CMV vector were co-transfected into different tumor cell lines and primary huamn gingival fibroblasts using Lipofectamine2000.The transcription activities of hTERT promoters were analyzed by dual-luciferase assay.Results：hTERT promoter and CWV enhancer were successfully cloned and the recombinant luciferase expression vectors were successfully constrcuted by restriction enzyme digestion and sequencing.Luciferase assay showed that the relatively transcriptional activities of wild hTERT promoter were 9.9% to 24.4% in different tumor cell lines and the activities increased in most modified hTERT promoters.Compared with wild hTERT promoter,the increase of luciferase activities were 1.3 to 4.9 fold in SV40 enhancer,4.0 to 42.8 fold in CWV enhancer,2.0 to 49.2 fold in CWV and SV40 double enhancer,0 to 5.5 fold in MMRE and 1.2 to 8.4 fold in MMRE combined with SV40 enhancer modified hTERT promoters.The transcriptional activities of the wild hTERT promoter and modified hTERT promoters were veried low in primary huamn gingival fibroblasts.Transcriptional activities of each hTERT promoter varied in different tumor cell lines.Conclusion：Compared with wild hTERT promoter,the transcriptional activities increase in most modified hTERT promoters.The hTERT promoter modified with CWV enhancer or SV40-CWV double enhancer has the highest transcriptional activities and might be used in targeted cancer gene therapy.