食管癌EC-1细胞株polβ启动子突变性转录活性分析  

Influence of promoter region ate-mutant of DNA polymerase β gene on its transcriptional activity in EC-1 cell

作　　者：李晓坤[1] 王欢[1] 杨妍[1] 李啸然[1] 赵国强[2] 董子明[3] 李月白[1] 

机构地区：[1]郑州大学基础医学院生物化学与分子生物学教研室,河南郑州450001 [2]郑州大学基础医学院微生物与免疫教研室,河南郑州450001 [3]郑州大学基础医学院病理与病生教研室,河南郑州450001

出　　处：《中国公共卫生》2011年第2期177-179,共3页Chinese Journal of Public Health

基　　金：河南省教育厅自然科学基础研究计划项目(2008A310017)

摘　　要：目的探讨食管癌细胞EC-1中DNA聚合酶β(DNA polymerase beta,polβ)基因启动子的突变对其转录活性的影响。方法提取正常永生化细胞293T和EC-1细胞基因组DNA,PCR扩增及测序分析,获得293T细胞野生型及EC-1细胞含-137位和-166位点G→A突变的突变型polβ启动子序列,构建polβ启动子萤光素酶报告基因重组质粒pGL3-neo-293T-polβ/promoter(野生型)和pGL3-neo-EC-1-polβ/promoter(突变型),重组质粒分别转染EC-1、Eca9706或293T细胞株,氨基糖苷类抗生素G418筛选,检测各组萤光素酶活性。结果野生型及突变型的polβ启动子片段正确插入萤光素酶报告基因载体pGL3-neo-enhancer,成功构建polβ启动子萤光素酶报告基因重组质粒;在3个细胞株中,野生型和突变型重组质粒萤光素酶活性均高于对照组,突变型高于野生型,差异均有统计学意义(P<0.001);突变型重组质粒在EC-1、Eca9706或293T细胞株中的萤光素酶活性值分别为(544 429.5±26 741);(505 683±26 661.1);(415 569.33±32 602.7),差异有统计学意义(P<0.05或P<0.001)。结论食管癌细胞EC-1中DNApolβ基因启动子-137位和-166位点G→A突变可增强其启动子活性;突变型polβ启动子报告基因载体在食管癌细胞中启动子活性较高,提示食管癌细胞中可能存在使其突变型polβ启动子活性增加的作用因子。Objective To investigate the influence of promoter region ate-mutant of DNA polymerase β gene（polβ） on its transcriptional activity in esophageal carcinoma cell line（EC-1）.Methods We extracted genomic DNA of normal immortalized cell 293T and EC-1 cell.The sequences of the wild-type polβ in 293T and mutant-type polβ with mutation at-137nt and-166nt（G→A） sites in EC-1 were obtained with polymerase chain reaction（PCR） and DNA sequence analysis.Then the luciferase reporter gene recombinant plasmids pGL3-neo-293T-polβ/promoter（wild-type） and pGL3-neo-EC-1-polβ/promoter（mutant-type） were built.The recombinant plasmids were transfected into EC-1,Eca9706 or 293T cells and screened by G418.The luciferase activity of each group was detected.Results The wild-type and mutant-type polβ promoters were inserted into pGL3-neo-enhancer in right direction.Control group（pGL3-neo-enhancer）,wild-type group and mutant group were transfected into EC-1,Eca9706 and 293T cells,respectively.The differences of luciferase activities between the wild and the control group,the mutant and the control group were significant（P0.001）.There was no significant difference between wild-type and control-type group（P 0.05） as well as between mutant EC-1,Eca9706 and 293T cells（P0.05,P0.001,P0.001）.Conclusion The mutation at-137nt and-166nt（G→A） of DNA polβ promoter in EC-1 can enhance the promoter activity.The promoter activity of the mutant DNA polβ promoter reporter gene vector is significantly higher in esophageal carcinoma cell than in immortalized normal cell 293T indicating that there may be some factors enhancing the mutant DNA polβ promoter activity in esophageal cancer cells.

关 键 词：DNA聚合酶β(polβ) 萤光素酶报告载体 启动子 突变 食管癌细胞 

分 类 号：R735.1[医药卫生—肿瘤]