线粒体基因组变异影响大肠癌进展及预后的作用机制研究

发布时间：2018-03-08 06:30

  本文选题：大肠癌　切入点：线粒体　出处：《第四军医大学》2017年博士论文　论文类型：学位论文

【摘要】：mtDNA的变异包括三大类,即线粒体拷贝数的变异、胚系突变的异质性转换及线粒体体细胞突变,mtDNA变异与肿瘤的进化演变密切相关。目前为止几乎在所有类型的肿瘤中都检测到mtDNA变异的存在。这些变异可引起细胞线粒体氧化胁迫,导致恶性循环。我参与的研究发现线粒体拷贝数升高与大肠癌的发病风险显著相关,而且mtDNA拷贝数与大肠癌分级分期及恶性进展也显著相关。在此基础上,本研究拟系统分析mtDNA变异与大肠癌进展及预后的关系,并深入探讨其作用机制。本研究分为以下三部分实验:第一部分:我们在120例大肠癌组织样本中检测mtDNA拷贝数和TFAM的表达情况,构建无mtDNA以及mtDNA拷贝数异常改变的细胞模型,全面检测细胞周期、细胞增殖、细胞凋亡、细胞侵袭迁移能力的变化,并通过裸鼠皮下成瘤实验检测mtDNA异常改变后细胞成瘤能力的改变,在此基础上我们还检测了不同mtDNA拷贝数对线粒体呼吸功能的影响。结果发现:(1)在微卫星稳定的大肠癌组织中mtDNA拷贝数异常升高,而在微卫星不稳定的癌组织中则相反,TFAM表达水平在两种微卫星状态的癌组织中也截然相反。(2)mtDNA拷贝数与TFAM表达水平具有显著相关性,且与大肠癌患者预后具有一致的趋势。(3)异常升高的mtDNA拷贝数可显著促进大肠癌细胞增殖、侵袭迁移,同时抑制细胞凋亡。(4)过表达TFAM使mtDNA拷贝数升高后,细胞成瘤能力增强。(5)增加mtDNA拷贝数可使细胞转移能力和增殖能力增强。(6)mtDNA拷贝数异常升高可显著促进细胞氧耗速率,促进细胞ROS和ATP产生,细胞膜电位降低。第二部分:我们通过对116例大肠癌组织mtDNA深度测序结果,结合公共数据库数据,系统分析比较了大肠癌组织中SNP shift数量、分布、功能及预后等。结果发现:(1)大肠癌/癌旁组织中共发现172个SNP shift突变位点,正常组织/血液中共发现75个SNP shift突变位点。(2)癌组织中以正向SNP shift为主。(3)癌组织中SNP shift突变以GA,TC转换形式为主。第三部分:我们通过对116例大肠癌组织mtDNA深度测序结果,结合公共数据库数据,系统分析比较了大肠癌组织中mtDNA突变数量、分布、功能及预后等。结果发现:(1)大肠癌组织中共发现110个mtDNA突变位点,平均每个肠癌患者包含0.95个突变。(2)按照大肠癌组织mtDNA突变位点位置,绘制肠癌mtDNA突变图谱。(3)癌组织中complex 1区域发生的突变占总体突变的百分比明显高于正常组织,而正常组织中D-loop区发生的突变占总体突变的百分比明显高于癌组织。(4)癌组织中以GA,TC的转换为主。本研究系统分析了线粒体基因组变异对大肠癌进展及预后的影响,并初步阐明了mtDNA拷贝数异常影响大肠癌进展及预后的作用机制,为系统研究线粒体基因组变异提供了实验基础,为推进临床大肠癌精准分子分型体系的构建提供了有益的思考。
[Abstract]:The variation of mtDNA consists of three categories, that is, mitochondrial copy number variation. The heterogeneity of blastocyte mutation and mitochondrial somatic mutation / mtDNA mutation are closely related to the evolution of tumor. Up to now, the existence of mtDNA mutation has been detected in almost all types of tumors. Mitochondrial oxidative stress, I participated in a study that found that increased mitochondrial copy numbers were significantly associated with the risk of colorectal cancer, and that mtDNA copy numbers were significantly associated with colorectal cancer grade, stage and progression. This study was intended to systematically analyze the relationship between mtDNA variation and the progression and prognosis of colorectal cancer. This study was divided into the following three parts: the first part: we detected the expression of mtDNA copy number and TFAM in 120 samples of colorectal cancer, and constructed a cell model without abnormal changes of mtDNA and mtDNA copy number. The changes of cell cycle, cell proliferation, cell apoptosis, cell invasion and migration were detected. The change of cell tumorigenesis ability after abnormal mtDNA changes was detected by subcutaneous tumorigenesis assay in nude mice. On this basis, we also examined the effects of different mtDNA copy numbers on mitochondrial respiratory function. The results showed that the mtDNA copy number increased abnormally in microsatellite stable colorectal cancer tissues. On the contrary, the expression of TFAM in microsatellite unstable cancer tissues was significantly correlated with the expression of TFAM in two kinds of microsatellite cancer tissues, and the number of copies of mtDNA was significantly correlated with the expression of TFAM in two kinds of microsatellite carcinomas, and the expression level of TFAM was significantly correlated with the expression of TFAM. The abnormal increase of mtDNA copy number could significantly promote the proliferation, invasion and migration of colorectal cancer cells, and inhibit the overexpression of TFAM, which resulted in the increase of mtDNA copy number. Increasing the copy number of mtDNA can enhance the ability of cell metastasis and proliferation. The abnormal increase of copy number of mtDNA can significantly promote the rate of cell oxygen consumption and the production of ROS and ATP. The second part: we analyzed and compared the number and distribution of SNP shift in colorectal cancer tissue by deep sequencing of mtDNA in 116 cases of colorectal cancer, combined with the data of common database, and compared the number and distribution of SNP shift in colorectal cancer tissue. Function and prognosis. The results showed that 172 SNP shift mutation sites were found in colorectal carcinoma / paracancerous tissues. A total of 75 SNP shift mutation sites were found in normal tissue / blood.) positive SNP shift was the main mutation of SNP shift in cancer tissues. In the third part, we analyzed the results of mtDNA deep sequencing in 116 colorectal cancer tissues. The number, distribution, function and prognosis of mtDNA mutations in colorectal cancer tissues were systematically analyzed and compared with common database data. The results showed that 110 mtDNA mutation sites were found in colorectal cancer tissues. An average of 0.95 mutations per patient with colorectal cancer.) according to the location of mtDNA mutation sites in colorectal cancer tissues, the percentage of mutations occurring in complex 1 region in colorectal cancer tissues was significantly higher than that in normal tissues, according to the location of mtDNA mutation loci in colorectal cancer tissues, and the percentage of mutations in complex 1 region in cancer tissues was significantly higher than that in normal tissues. However, the percentage of mutations in D-loop region in normal tissues was significantly higher than that in cancer tissues. In this study, the effect of mitochondrial genome mutation on the progression and prognosis of colorectal cancer was systematically analyzed. The mechanism of the abnormal copy number of mtDNA affecting the progression and prognosis of colorectal cancer was elucidated, which provided the experimental basis for systematic study of mitochondrial genome variation, and provided useful thoughts for the construction of accurate molecular typing system for clinical colorectal cancer.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.34
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本文编号：1582795