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李晓坤, 王欢, 杨妍, 李啸然, 赵国强, 董子明, 李月白. 食管癌EC-1细胞株pol启动子突变性转录活性分析[J]. 中国公共卫生, 2011, 27(2): 177-179. DOI: 10.11847/zgggws2011-27-02-25
引用本文: 李晓坤, 王欢, 杨妍, 李啸然, 赵国强, 董子明, 李月白. 食管癌EC-1细胞株pol启动子突变性转录活性分析[J]. 中国公共卫生, 2011, 27(2): 177-179. DOI: 10.11847/zgggws2011-27-02-25
LI Xiao-kun, WANG Huan, YANG Yan, . Influence of promoter region ate-mutant of DNA polymerase gene on its transcriptional activity in EC-1 cell[J]. Chinese Journal of Public Health, 2011, 27(2): 177-179. DOI: 10.11847/zgggws2011-27-02-25
Citation: LI Xiao-kun, WANG Huan, YANG Yan, . Influence of promoter region ate-mutant of DNA polymerase gene on its transcriptional activity in EC-1 cell[J]. Chinese Journal of Public Health, 2011, 27(2): 177-179. DOI: 10.11847/zgggws2011-27-02-25

食管癌EC-1细胞株pol启动子突变性转录活性分析

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

  • 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。

     

    Abstract: Objective To investigate the influence of promoter region ate-mutant of DNA polymerase β gene(pol β) on its transcr iptional activity in esophageal carcinoma cell line (EC-1).Methods We extracted genomic DNA of normal mimortalized cell 293T and EC-1 cell.The sequences of the wildtype pol β in 293T and mutanttype pol β with mutation at -137nt and -166nt(G→A) sites in EC-1 were obtained with polyme rase chain reaction(PCR) and DNA sequence analysis.Then the luciferase reporter gene recombinant plasm ids pGL 3-neo-293T-pol β/promoter(wild-type) and pGL 3-neo-EC1-pol β/promoter(mutant-type) were built.The recom binant plasmids were transfected into EC-1,Eca9706 or 293T cells and screened by G418.The lucife rase activity of each group was detected.Results The wild-type and mutant-type pol β promoters were inserted in to pGL3-neo-enhancer in right direction.Control group (pGL 3-neo-enhancer),wild-type group and mutant group were transfected into EC-1,Eca9706 and 293T cells,respectively.The differences of luciferase activitie s between the wild and the control group,the mutant and the control group were significant(P< 0.001).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(P < 0.05,P < 0.001,P < 0.001).Conclusion The mutation at -137nt and -166nt(G→A) of DNA pol β promoter in EC-1 can enhance the promoter activity.The promoter activity of the mutant DNA pol β promoter reporter gene vector is significantly higher in esophageal carcinoma cell than in mimorta lized normal cell 293T indicating that there maybe some factors enhancing the mutant DNA pol β promoter activity in esophageal cancer cells.

     

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