核内PTEN通过阻遏Ku70与DNA的结合下调非同源末端连接效率

发布时间：2018-01-30 04:47

本文关键词： PTEN 同源重组非同源末端连接 Ku70 双链断裂修复　出处：《大连医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景:PTEN(phosphatase and tensin homologue,磷酸酶与张力蛋白同源物)作为一个抑癌蛋白,过去最为人们所熟知的是其磷酸酶活性。凭借这一性质,PTEN在细胞质中能够拮抗PI3K(phosphoinositide 3-kinase,磷脂酰肌醇-3-激酶)-PKB(protein kinase B,蛋白激酶B)通路,起到抑制细胞生长增殖及存活的作用。除了阻遏PI3K通路之外,具有磷酸酶活性的PTEN蛋白在线粒体氧化磷酸化等多种生理过程中也发挥着重要作用。随着PTEN研究的深入,该蛋白在细胞核内维持基因组稳定性方面的作用受到广泛关注。基因组稳定性与DNA结构的完整性密切相关,活性氧及电离辐射等因素攻击细胞DNA时,常常使细胞DNA链受到损伤,其中双链断裂损伤是危害性最大的一类。DNA双链断裂损伤修复的途径主要有两条,分别为同源重组修复方式和非同源末端连接通路。同源重组修复发生在S期或G2期,以未受损的姐妹染色单体DNA为模板,在多种蛋白的作用下进行精确修复。而非同源末端连接并不需要同源链做为模板,因此,修复后往往存在DNA序列的改变,这类修复方式并不保真。目前,虽然认为PTEN在双链断裂修复中发挥重要作用,但在这两种修复通路中的具体作用机制仍不清楚。目的:本研究主要探索了PTEN在同源重组修复途径和非同源末端连接通路中所起的作用以及分子机制。癌症的发生往往伴随着PTEN功能的缺失,因此,对于该蛋白在DNA双链断裂修复方式中作用的研究能够揭示PTEN缺失型癌症独特的DNA修复方式,从而为癌症病人的临床诊疗方案提供更多的理论支持及新思路。方法:重叠延伸PCR法用于构建不同PTEN的突变体。单细胞凝胶电泳实验用于检测不同细胞发生DNA双链断裂损伤后的修复能力。免疫荧光实验用于检测磷酸化H2AX灶点数量。Western blot用于检测各细胞系中PTEN蛋白及非同源末端连接、同源重组修复通路中相关蛋白的表达。基于pUC19质粒的不相容末端连接法以及非同源末端连接质粒报告系统法用于检测细胞的非同源末端连接效率。电泳迁移率实验用于检测Ku70复合物与生物素标记探针模拟的DNA双链断裂处的结合能力。染色质免疫共沉淀实验用于检测细胞内Ku70蛋白与DNA双链断裂损伤处的结合能力。MTT实验用于检测携带不同PTEN突变体的细胞对各类DNA损伤药物的敏感性。结果:在缺失内源性PTEN的人乳腺癌细胞系BT549中,我们分别稳定转染了野生型PTEN、磷酸酶活性位点突变PTEN以及小泛素化位点突变PTEN。单细胞凝胶电泳实验以及磷酸化H2AX的检测证明PTEN促进了细胞DNA双链断裂修复的进程,且它的这一功能在PTEN磷酸酶活性受损后并不降低,而该作用在PTEN小泛素化位点突变不能入核后降低。Western blot检测携带不同PTEN突变体的细胞遭受辐射处理后Rad51表达情况,结果证明细胞核内存在PTEN时,细胞的同源重组修复效率较高。基于pUC19质粒的不相容末端连接法以及非同源末端连接质粒报告系统法证实核内PTEN阻遏了非同源末端连接通路的修复效率。进一步实验发现,虽然非同源末端连接途径的关键蛋白Ku70在各细胞系中表达量没有显著差异,但电泳迁移率实验结果表明核内PTEN存在的情况下,结合到生物素标记的探针上的Ku70复合体相较于核内PTEN缺失的实验组减少了两到三倍。染色质免疫共沉淀的结果也证实了核内PTEN阻遏了Ku70与DNA的结合。在另一种存在内源性PTEN的乳腺癌细胞系MDA-MB-231细胞中干扰PTEN后进行电泳迁移率实验证实PTEN缺失后Ku70与探针结合能力显著提高。MTT实验证实核内PTEN缺失导致细胞对各类DNA损伤药物更为敏感。结论:我们的实验结果证实,在细胞遭受DNA双链断裂损伤时,核内PTEN通过阻遏Ku70与DNA断裂处的结合而下调保真性较差的非同源末端连接修复途径,同时通过上调同源重组修复通路中的关键因子Rad51蛋白的表达而促进同源重组修复途径,从而维持了DNA的稳定性。并且,PTEN的这一作用依赖于其在细胞核内的定位,而与磷酸酶活性无关。
[Abstract]:Background: PTEN (phosphatase and tensin homologue, phosphatase and tensin homolog) as a tumor suppressor protein, the past is best known for its phosphatase activity. By virtue of this property, PTEN can antagonize PI3K in the cytoplasm (phosphoinositide 3-kinase, phosphatidylinositol -3- kinase -PKB (protein) kinase B protein. B kinase) pathway, inhibit cell growth and survival proliferation. In addition to blocking PI3K pathway with phosphatase activity of PTEN protein also plays an important role in various physiological processes such as mitochondrial oxidative phosphorylation. With the deep research of PTEN, the protein in maintaining genomic stability in the nuclei of the role of attention the integrity of the genome. The stability and the structure of DNA is closely related to the DNA attack of active oxygen and cell factors such as radiation, often make the DNA chains of cells are damaged, which Double strand breaks are the two main ways of the biggest harm of a kind of.DNA double strand break repair, homologous recombination repair respectively and non homologous end joining pathway. Homologous recombination repair occurs in S or G2, with intact sister chromatid DNA as template, precise repair in a variety of proteins. The non homologous end joining does not require homologous strand as a template, therefore, there are DNA sequence changes after repair, the repair is not fidelity. At present, although that PTEN play an important role in DSB repair, but the specific mechanism of action in the two repair pathway in is still not clear. Objective: This study explored the PTEN in homologous recombination repair pathway and non homologous end joining pathway plays the role and molecular mechanism. The occurrence of cancer is often accompanied by a lack of PTEN function because of this, the Study on the role of protein in the repair of DNA double strand breaks in the repair can reveal the deletion of PTEN cancer DNA unique, so as to provide new ideas and theoretical support for more clinical diagnosis and treatment of cancer patients. Methods: PCR method for overlap extension PTEN. The mutant construct different single cell gel electrophoresis for detecting different cells have ability to repair DNA double strand breaks after injury. Immunofluorescence assay was used to detect the phosphorylation of H2AX on focal point number of.Western blot for the detection of PTEN protein in various cell lines and non homologous end joining, homologous recombination repair pathway related protein expression. PUC19 plasmid incompatibility end joining method and non homologous end joining. Report system method for detecting cell non homologous end joining efficiency. Based on the electrophoretic mobility shift assay for detection of Ku70 complex and labeled probe. The binding ability of DNA double strand breaks. The chromatin immunoprecipitation for detection of intracellular Ku70 protein and DNA double strand breaks the binding capacity of.MTT experimental sensitivity for detecting PTEN mutant cells carrying different drugs for various types of DNA damage by coprecipitation experiments. Results: in human breast cancer cell line BT549 deletion of endogenous PTEN we are in stable transfection of wild-type PTEN, PTEN mutation and phosphatase activity of sumoylation site mutation PTEN. of single cell gel electrophoresis and detection of phosphorylated H2AX showed that PTEN promoted cell DNA double strand break repair process, and the function of it in the phosphatase activity of PTEN after damage is not reduced, and the role of PTEN in small ubiquitin mutant Rad51 expression after.Western cells exposed to radiation treatment reduced blot detection with a different PTEN mutants can not enter the nucleus, results show that The presence of PTEN in the nucleus of cells, homologous recombination repair efficiency is high. The repair efficiency of pUC19 plasmid incompatibility end joining method and non homologous end joining plasmid was confirmed in nuclear PTEN reporting system to prevent non homologous end joining pathway. Based on further experiments found that although the non homologous end joining pathway of Ku70 protein in the cell lines expression had no significant difference, but the electrophoretic mobility of the experimental results indicate the presence of nuclear PTEN, Ku70 complex binding to the biotinylated probe on compared to the experimental group in the nucleus PTEN deletion was reduced by two to three times. Chromatin immunoprecipitation results also confirmed that the nuclear PTEN the combination of Ku70 and repressor of DNA. The interference of PTEN in breast cancer cell lines MDA-MB-231 cells another endogenous PTEN in electrophoretic mobility after confirmed after deletion of PTEN and Ku70 probe With the ability to significantly improve the.MTT experiments confirmed that the nuclear PTEN lack of cells are more sensitive to various types of DNA damage drugs. Conclusion: Our results demonstrate that in cells exposed to DNA double strand breaks, non homologous end joining repair pathway in nuclear PTEN by combining Ku70 and DNA repression at fracture and lowered the fidelity of the poor at the same time, the expression of Rad51 protein by upregulating the homologous recombination repair pathway is the key factor in the promotion of homologous recombination repair pathway, so as to maintain the stability of DNA. Moreover, the effect of PTEN depends on its position in the nucleus, but has nothing to do with the phosphatase activity.

【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R73;Q78

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