胰腺癌全基因组甲基化差异分析及重要功能基因的临床应用价值研究

发布时间：2018-06-10 18:27

本文选题：胰腺癌 + 基因芯片　；参考：《中国人民解放军医学院》2015年博士论文

【摘要】：胰腺癌恶性程度高,早期诊断困难,缺乏有效治疗手段。迫切需要高效诊断标志物及疗效确切的靶向治疗药物。DNA甲基化在胰腺癌发生、发展过程中作用重要,基因甲基化研究可能成为胰腺癌诊断及治疗的突破。研究应用Human Methylation 450K芯片对癌旁组织、胰腺癌组织、胰腺癌血液检测,通过芯片扫描、数据分析得出异常甲基化位点及基因。选取代表基因验证及功能研究,以期发现新的胰腺癌诊断及治疗标志物。第一部分：通过分析比较胰腺癌组织与癌旁组织的异常甲基化位点,得到胰腺癌组织全基因组甲基化图谱。主要结果：1、胰腺癌与癌旁基因甲基化程度在基因组范围内线性相关；2、胰腺癌基因组甲基化CpG位点数目远低于非甲基化CpG位点数目,启动子区则以高甲基化为主；3、异常甲基化基因GO和Pathway分析,得到差异基因GO和Pathway信号通路；4、筛选出高频甲基化基因参与的GO通路和Pathway信号通路；5、按甲基化程度分析,获得启动子区主要高甲基化基因及低甲基化基因；6、获得了异常甲基化基因参与的多条信号通路图。第二部分：癌组织和癌旁组织(CvsB)比较、血液和癌旁组织(AvsB)比较,对各自结果进行再比较(CvsB)VS(AvsB),得到异常甲基化CpG位点集(以下简称overlap集),对overlap集分析,得到胰腺癌血液DNA甲基化图谱及甲基化修饰模式。主要结果：1、胰腺癌血液与癌旁的异常甲基化位点为线性关系；2、聚类分析表明,A、B、C组内差异不明显,而组间差异明显,体现了研究对象的代表性；3、获得了异常甲基化CpG位点分布和基因组定位；4、筛选胰腺癌血液异常甲基化基因,得到每个基因包含的差异CpG点信息；5、得到了胰腺癌血液启动子区异常甲基化基因的甲基修饰模式；6、启动子区重要异常甲基化基因GO分析和pathway分析；7、“胰腺癌KEGG信号通路”分析,发现启动子区重要异常甲基化基因参与胰腺癌的发生、发展。第三部分：选取MADIL1基因进行血液学飞行质谱验证,探讨了其作为胰腺癌诊断标志物的可行性。第四部分：选取ASS1基因为研究对象,明确了ASS1作为精氨酸合成的限速基因在胰腺癌细胞株之间甲基化程度差异、表达差异及作用机制,探讨了精氨酸剥夺对ASS1的影响,发现了甲基化抑制剂5-AZA的作用机制,探讨了ADI精氨酸剥夺抑制胰腺癌细胞迁移、侵袭以及胰腺癌细胞聚集的分子机制。为胰腺癌“饥饿”治疗奠定理论基础。
[Abstract]:Pancreatic cancer has a high degree of malignancy, difficult early diagnosis and lack of effective treatment. There is an urgent need for highly effective diagnostic markers and targeted therapeutic drugs. DNA methylation plays an important role in the development of pancreatic cancer. The study of gene methylation may be a breakthrough in the diagnosis and treatment of pancreatic cancer. Human Methylation 450K microarray was used to detect the blood of paracancerous tissues and pancreatic cancer. The abnormal methylation sites and genes were obtained by microarray scanning and data analysis. To find new diagnostic and therapeutic markers of pancreatic cancer, we selected representative gene validation and functional study. Part one: by analyzing and comparing the abnormal methylation sites between pancreatic cancer tissues and paracancerous tissues, the whole genome methylation map of pancreatic cancer tissue was obtained. The main results were as follows: 1. The methylation degree of pancreatic cancer was linearly correlated with the degree of para-oncogene methylation. The number of methylated CpG loci in pancreatic cancer genome was much lower than that of unmethylated CpG loci. Hypermethylation was dominant in promoter region, abnormal methylation gene go and Pathway were analyzed, and differential gene go and Pathway signaling pathway 4 were obtained. Go pathway and Pathway signaling pathway involved by high frequency methylation gene were screened out. The main hypermethylation genes and low methylation genes in promoter region were obtained, and several signal pathway diagrams of abnormal methylation genes were obtained. The second part: the comparison of CvsBs in cancer tissue and adjacent tissue, and the comparison between blood and paracancerous tissue (AvsB), and the comparison of their results. The abnormal methylated CpG locus (hereinafter referred to as overlap set) was obtained, and the overlap set was analyzed. Blood DNA methylation patterns and methylation modification patterns of pancreatic cancer were obtained. The main results were as follows: 1. The abnormal methylation sites in blood of pancreatic cancer were linear. Cluster analysis showed that there was no significant difference in the blood methylation sites between the two groups, but there was a significant difference between the two groups. We obtained abnormal methylation CpG locus distribution and genomic localization 4, and screened abnormal methylation gene in blood of pancreatic cancer. The different CpG site information of each gene was obtained. The methylation pattern of abnormal methylation gene in blood promoter region of pancreatic cancer was obtained. The important abnormal methylation gene in promoter region was analyzed by go and pathway, and the KEGG signaling pathway of pancreatic cancer was analyzed. Important abnormal methylation genes in promoter region were found to be involved in the development of pancreatic cancer. Part three: MADIL1 gene was selected to be validated by hematological FMS, and the feasibility of MADIL1 as a diagnostic marker for pancreatic cancer was discussed. Part four: ASS1 gene was selected as the research object. The difference of methylation, expression and mechanism of ASS1 as a rate-limiting gene for arginine synthesis between pancreatic cancer cell lines was determined, and the effect of arginine deprivation on ASS1 was discussed. The mechanism of methylation inhibitor 5-AZA was found, and the molecular mechanism of ADI arginine deprivation in inhibiting the migration, invasion and cell aggregation of pancreatic cancer cells was explored. To lay a theoretical foundation for the treatment of pancreatic cancer hunger.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.9

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