不同截短型癌基因 AEG －1启动子活性分析

摘要

目的：分析不同截短型癌基因 AEG －1启动子活性，证实与 E6／E7表达相关的转录调控因子对AEG －1启动子活性的影响。方法：以宫颈癌 HeLa 细胞基因组 DNA 为模版，扩增 AEG －1启动子全长序列（－2710／－491），并应用本实验室改造的启动子活性研究专用绿色荧光蛋白报告载体构建 pGL3－basic －EGFP／AEG －1质粒，通过转染 HeLa 细胞及血管内皮细胞 ECV304检测启动子活性。同时，根据 AEG －1启动子序列上与 E6／E7作用相关的转录调控因子结合位点位置，设计不同截短型 AEG －1启动子序列引物，应用 pGL3－basic －EGFP 载体构建不同截短型 AEG －1启动子载体，并分别转染细胞检测启动子活性。结果：AEG －1启动子全长序列 pGL3－basic －EGFP／AEG －1载体转染 HeLa 细胞可见明显绿色荧光蛋白表达，而在血管内皮细胞 ECV304中表达为痕量。包含不同转录调控因子 C －Myc、SP1、NF －κB、E2 F 结合位点的不同截短型 AEG －1启动子载体在肿瘤细胞中有活性但存在差异。结论：AEG －1启动子为肿瘤特异性启动子，含有与 E6／E7表达相关的转录调控因子 NF －κB、E2 F 结合位点的启动子序列为影响 AEG －1基因启动子活性的关键区域。%Objective:To analyze the activity of truncated oncogene AEG -1 promoter and to confirm the effects of E6 /E7 related transcription regulatory factors on the AEG -1 promoter activity.Methods:The full length sequences of AEG -1 gene promoter(-2710 /-491)were amplified from HeLa cell genome DNA,and then cloned into pGL3-basic -EGFP vectors to constructe the promoter report plasmid of pGL3 -basic -EGFP/AEG -1 that was then transfected into HeLa and ECV304 cells to detect the AEG -1 promoter tumor specific activity.Meanwhile,differently truncated region of AEG -1 promoters were designed according to the transcriptional regulatory factors position on promoter sequences,and the activity were separately detected with the method described as above.Results:Strong green fluorescence could be observed in HeLa cells,but not in ECV304 cells when transfected with pGL3 -basic -EGFP/AEG -1 vector.The activities for differently truncated region of AEG -1 promoters sequences with transcrip-tional regulation factors C -Myc,SP1,NF -κB,E2 F binding sites had significant difference signals of green fluores-cence.Conclusion:AEG -1 gene promoter is a tumor specific activity promoter.The promoter sequences which have transcriptional regulation factor NF -κB,E2 F,all of which are related to E6 /E7 function influences the activity of the promoter sequences.