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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 effect of mutation of DNA polymerase beta (polβ) gene promoter on the transcription activity of esophageal cancer cell EC-1. Methods The genomic DNA of 293T and EC-1 cells from normal immortalized cells was extracted and sequenced. The mutant polβ was obtained from wild-type 293T cells and G-A mutant containing -137 and -166 sites in EC-1 cells The polβ promoter luciferase reporter gene recombinant plasmid pGL3-neo-293T-polβ / promoter (wild type) and pGL3-neo-EC-1-polβ / promoter EC-1, Eca9706 or 293T cell lines, aminoglycoside antibiotics G418 screening, detection of each group of luciferase activity. Results The wild-type and mutant polβ promoter fragments were correctly inserted into the luciferase reporter gene vector pGL3-neo-enhancer, and the polβ promoter luciferase reporter gene recombinant plasmid was successfully constructed. Among the three cell lines, wild type and The luciferase activity of the mutant recombinant plasmid was higher than that of the control group, and the mutant type was higher than that of the wild type (P <0.001). The mutant recombinant plasmid was found in EC-1, Eca9706 or 293T cell lines The values of luciferase activity were (544 429.5 ± 26 741), (505 683 ± 26 661.1) and (415 569.33 ± 32 602.7), respectively. The difference was statistically significant (P <0.05 or P <0.001). CONCLUSION: The promoter of DNApolβ gene promoter at position -137 and -166 in esophageal carcinoma EC-1 cells may enhance its promoter activity. The reporter gene of mutant polβ promoter has higher promoter activity in esophageal cancer cells, Suggesting that esophageal cancer cells may have mutation-type polβ promoter activity increased factor.