雌二醇上调人DNA错配修复基因hMLH1作用机制的初步研究

发布时间：2018-06-28 08:42

  本文选题：错配修复基因 + hMLH1　； 参考：《第三军医大学》2012年硕士论文

【摘要】：研究背景及目的 人DNA错配修复（mismatch repair, MMR）系统在维持基因组稳定性中起着关键作用。MMR功能障碍导致的微卫星不稳定（microsatellite instability,MSI）与许多恶性肿瘤的发生有密切关系。大部分遗传性非息肉病性结直肠癌（hereditary nonpolyposis colorectal cancer, HNPCC）由MMR基因种系突变引起。此外，一部分散发性结直肠癌也与MMR功能障碍相关。hMLH1是体内最重要的MMR基因之一。前期研究发现，正常人血清雌激素（Estradiol，E2）水平与结肠上皮细胞中hMLH1基因表达水平呈正相关。细胞实验证实，E2能上调结肠癌细胞株COLO205的hMLH1基因表达量，并且这种上调作用很可能发生在转录水平。然而，E2如何调节错配修复基因hMLH1表达的具体机制尚不清楚。本研究旨在构建含hMLH1启动子片段的荧光素酶报告基因载体，检测其在HEK293和LoVo细胞株中E2诱导下的转录活性。 研究方法 1、根据UCSC（www.geome.ucsc.edu）数据库确定hMLH1基因转录起始位点，用PCR方法克隆hMLH1启动子序列（-1953/+53），，定向插入到双荧光报告基因载体pGL3-Basic，抽提质粒并经双酶切和测序鉴定。构建好的含hMLH1启动子的真核表达质粒命名为pGL3-Promoter-luc。 2、用瞬时转染的方法将pGL3-Promoter-luc、阴性对照（pGL3-Basic）和阳性对照（pGL3-Control）分别与内参质粒（pRL-SV40）共转入HEK293和LoVo细胞。检测不同作用时间和不同剂量的E2对报告基因荧光素酶活性值的影响，以及E2-BSA和ICI182.780对hMLH1启动子转录活性的影响。 3、运用Western Blot法检测HEK293和LoVo细胞中hMLH1的相对表达量。 结果 1、构建的含hMLH1启动子的重组质粒经酶切和测序鉴定,插入的核苷酸序列与UCSC数据库中hMLH1启动子区序列完全吻合，说明报告基因重组质粒构建成功。 2、报告基因荧光素酶活性对E2存在一定的剂量依赖和时间依赖关系。10~(-9)mol/L的E2处理24小时后，报告基因荧光素酶活性值增强最明显（n=3，P0.01），而这种效应能被雌激素受体拮抗剂ICI182.780抑制。E2-BSA对目的基因的上调作用不如E2显著。 3、运用Western Blot检测HEK293和LoVo细胞株内源性hMLH1基因蛋白表达量，转染pRST7-ERβ组高于对照组（n=3，P0.01）。结论 E2能上调HEK293和LoVo细胞错配修复基因MLH1的表达，并能显著增强由hMLH1启动子序列引导的荧光素酶表达活性，说明hMLH1启动子序列中存在与E2相关的调控序列，且ERβ在此过程中起到重要作用。该报告基因载体为进一步明确参与调控的顺式作用元件和转录因子奠定了实验基础。
[Abstract]:Background and objective Human DNA mismatch repair (mismatch repair,) system plays a key role in maintaining genomic stability. The microsatellite instability (microsatellite instability) caused by MMR dysfunction is closely related to the occurrence of many malignant tumors. Most hereditary nonpolyposis colorectal cancer (hereditary nonpolyposis colorectal cancer, HNPCC) is caused by mutation of MMR gene. In addition, some sporadic colorectal cancer is also associated with MMR dysfunction. HMLH1 is one of the most important MMR genes in vivo. Previous studies showed that the level of serum estradiol E _ 2 (E _ 2) was positively correlated with the expression of hMLH1 gene in colonic epithelial cells. Cell experiments confirmed that E _ 2 could up-regulate the expression of hMLH1 gene in colon cancer cell line COLO205, and this up-regulation may occur at the transcription level. However, it is unclear how E2 regulates the expression of mismatch repair gene hMLH1. The aim of this study was to construct a luciferase reporter gene vector containing hMLH1 promoter fragment and to detect its transcriptional activity induced by E2 in HEK293 and LoVo cell lines. Methods 1.According to the UCSC (www.geome.ucsc.edu) database, hMLH1 promoter sequence (-1953 / 53) was cloned and inserted into the double fluorescent reporter gene vector pGL3-Basic. the plasmid was extracted and identified by double enzyme digestion and sequencing. The constructed eukaryotic expression plasmids containing hMLH1 promoter were named pGL3-Promoter-Luc.2. PGL3-Promoter-luc, pGL3-basic and pGL3-Control were co-transfected into HEK293 and LoVo cells with pRL-SV40, respectively. The effects of different time and dose of E2 on luciferase activity of reporter gene and the transcriptional activity of E2-BSA and ICI182.780 on hMLH1 promoter were detected. 3 the relative expression of hMLH1 in HEK293 and LoVo cells was detected by Western Blot. Results 1. The recombinant plasmid containing hMLH1 promoter was identified by restriction endonuclease digestion and sequencing. The inserted nucleotide sequence was consistent with the sequence of hMLH1 promoter in UCSC database. The results showed that the reporter gene recombinant plasmid was successfully constructed. 2. The luciferase activity of the reporter gene had a dose-dependent and time-dependent relationship with E2 for 24 hours after treatment with E2 of 10 ~ (-9) mol / L. The luciferase activity of the reporter gene was increased most significantly (nng3, P0.01), and this effect was inhibited by estrogen receptor antagonist ICI182.780. E2-BSA was not as up-regulated as E2. 3. Western Blot was used to detect the effect in HEK293 and LoVo cell lines. Protein expression of hMLH1 gene, The transfection of pRST7-ER 尾 group was higher than that of control group (nrST7-ER 尾 group). Conclusion E2 can up-regulate the expression of mismatch repair gene MLH1 in HEK293 and LoVo cells, and enhance the luciferase expression activity guided by hMLH1 promoter, indicating that there are E2 related regulatory sequences in hMLH1 promoter sequence. And ER 尾 plays an important role in this process. The report gene vector lays the experimental foundation for further clarifying the cis-acting elements and transcription factors involved in regulation.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R346

【参考文献】

相关期刊论文 前2条

1 陆晓娟;余东亮;王佳锌;潘笑露;金鹏;李世荣;盛剑秋;;雌激素对结肠癌细胞株COLO205错配修复基因表达的影响[J];细胞与分子免疫学杂志;2011年07期

2 牟韶娇;盛剑秋;谢惠;金鹏;陆晓娟;高巍;;雌激素对结肠细胞错配修复活性的影响[J];胃肠病学和肝病学杂志;2011年05期

本文编号：2077406