中国科学院昆明动物研究所机构知识库

癌症是一个世界性的公共健康问题，是全球最主要的致死原因之一。由于人口增长以及老龄化问题的加剧，由癌症导致的经济负担也在快速增长，造成了严重的社会问题。现代人的生活方式包括吸烟、过度饮酒、不良的饮食、缺乏身体锻炼以及生育方式的改变等则进一步增加了癌症的风险。癌症是一种复杂的疾病，异常的基因表达与各种肿瘤特性相关。在肿瘤发生过程中，癌基因通常被激活，而肿瘤抑制基因失活。研究发现有多种不同的基因表达调控机制，包括遗传学和表观遗传学变化。近年来，癌症表观遗传学领域吸引了研究人员越来越多的关注，研究表明表观遗传学在癌症发展中扮演越来越重要的角色，与肿瘤分类、治疗以及预后密切相关。DNA甲基化作为最常见的表观遗传修饰，是调节基因表达和染色质结构的桥梁，在调节基因和转座子沉默，基因组印记和X染色体失活中发挥关键作用。研究已经表明，基因启动子区域的DNA高甲基化会导致基因表达被抑制，而低甲基化会导致基因表达上调。通过基因编辑技术下调目标区域的基因启动子区域的DNA甲基化水平可以上调基因表达。在癌症中，DNA甲基化异常通常调节那些与细胞生长、增殖、分化和凋亡等过程相关的基因的表达水平。异常的DNA甲基化被证明是一个癌症细胞共同的特征。近期的研究通过泛肿瘤的分析发现可以依据DNA甲基化水平而把原本来源于不同组织类型的肿瘤归到一个新的肿瘤类型中，这项研究为癌症分类、诊断以及预后和治疗提供了新的策略。然而，癌症是高度异质性的，目前还不清楚不同癌症类型中是否存在共同的DNA甲基化模式或者DNA甲基化调控的基因表达模式。我们通过对12种实体瘤的DNA甲基化调控的基因表达模式进行分析，发现肿瘤中总体呈现出DNA高甲基化状态，相比于表观激活基因（DNA低甲基化且高表达基因），在12种肿瘤中均发现更多的表观沉默基因（DNA高甲基化且低表达基因），并且通过对表观沉默基因的信号通路富集分析以及基因相互作用网络分析都发现大部分钙信号通路基因呈现出高甲基化低表达状态。此外，基因表达受到DNA甲基化调控的这些基因多位于钙信号通路的关键节点上，包括控制钙流量的钠离子-钙离子交换器和G蛋白偶联受体等。利用这些异常的钙信号通路基因的DNA甲基化水平可有效的区分开肿瘤样本和正常对照样本，尤其需要注意的是我们的结果与Raynal 等人关于钙信号通路的研究结果也比较吻合，提示靶向钙信号通路基因的DNA甲基化水平确实可能作为肿瘤靶向治疗的新方向，我们的工作可以为后续关于肿瘤钙信号通路的研究特别是基于钙信号通路的药物开发研究提供支持。另一方面，我们想探究在基因表达层面以及DNA甲基化层面可以作为判断肿瘤分期及预后好坏与否的分子标记。鉴于此，我们从TCGA下载了11种肿瘤的基因表达数据、DNA甲基化数据和临床数据，通过与正常对照相比，找出肿瘤中差异表达的基因，之后对这些基因与肿瘤分期进行相关性分析，找出肿瘤分期相关的差异表达基因及DNA甲基化位点，最后再对这些基因与肿瘤预后进行分析最终找出与肿瘤分期相关并可作为肿瘤预后marker的差异表达基因和DNA甲基化位点，同时我们对这些基因进行GO的信号通路富集分析以探讨这些基因潜在参与的生物学通路。结果发现：在基因表达层面，与肿瘤分期正相关的通路主要是细胞周期以及DNA复制通路等，预后分析也发现细胞周期通路上的一些基因包括BUB1等可作为肿瘤预后的分子标记，与肿瘤分期呈负相关关系的通路主要包括细胞粘附和三羧酸循环通路；在DNA甲基化层面，有一些信号通路上的基因的DNA甲基化位点与肿瘤分期相关，包括钙信号通路、粘着斑通路、细胞骨架调控等信号通路。并且预后分析也发现这些基因与肿瘤预后相关。总之，本研究揭示：在12种实体肿瘤中呈现DNA高甲基化状态，并且这些高水平的DNA甲基化主要通过调控钙信号通路及细胞粘附通路相关的基因来发挥作用，这些基因位于钙信号通路的一些关键节点上，调控着细胞钙流量；在11种实体肿瘤中，发现一些与肿瘤分期及预后相关的基因，这些基因与细胞周期及DNA复制等过程相关，同时这些基因会随着肿瘤分期进行表达不断上升，其表达水平的高低可作为预测肿瘤患者生存率的分子标记，同时，一些DNA甲基化位点与肿瘤分期及预后相关，这些基因与钙信号通路、粘着斑通路、细胞骨架调控通路等相关，其甲基化水平亦可作为预测肿瘤患者生存率的分子标记。我们的研究发现了跨肿瘤的DNA高甲基化调控着钙信号通路基因，并且钙信号通路基因上的一些DNA甲基化位点与肿瘤的分期及预后相关。上述研究为采用生物信息学方法对肿瘤大数据研究进行跨肿瘤的基因表达调控，此外还结合肿瘤临床数据，探究肿瘤发生发展过程中的规律，以期为肿瘤诊断、发病机理、治疗等方面的进一步研究提供了理论支持，为将来的肿瘤靶向治疗和肿瘤精准医疗提供些许帮助。 

Cancer is a worldwide public health problem, which is one of the leading causes of death in the world.Due to population growth and human aging population increase, economic burden caused by cancer is fast-growing, and caused serious social problems.Modern lifesytle including smoking, drinking alcohol, poor diet, lack of physical exercise and change of fertility further increases the burden of cancer.Cancer is a complex disease, abnormal gene expression incancer during tumorigenesis linked to cancer features. Cancer related oncogenesareusually activated, and tumor suppressor genesare suppressed or inactivated. There are many different ways to regulategene expression, including genetic and epigenetic changes.In recent years, the field of cancer epigenetics has attracted more and more attentions, previous studies have shown that epigenetics plays an important role in cancer development, related with tumor classification, treatment and prognosis.As one of the most common epigenetic modifications, DNA methylation is served as the bridge between gene expression and chromatin structure. As the key factors, DNA methylation couldregulate gene and transposon silencing, genomic imprinting and X chromosome inactivation. Lots of studies have shown that high DNA methylation in the promoter region of the gene often lead to gene expression inhibition, and low methylation lead to gene expression activation. Recent study found demethylation of target area in the gene could activate gene expression.Abnormal DNA methylation in cancer usually regulate gene expression level related those processes such as cell growth, proliferation, differentiation and apoptosis related.Abnormal DNA methylation was proved to be a common characteristics in cancer cell. Recent research suggested that cancers come from different tissues origin could be divide into a new cancer type according to the level of DNA methylation in cancer, which is helpful for cancer classification, diagnosis and prognosis and treatment. Cancer is so heterogeneous, it is unclear whether there is common DNA methylation pattern or pattern of DNA methylation regulation gene expressionexisted in different cancer types.We analyzed the data of 12 solid tumor DNA methylation and gene expression, we found that DNA hypermethylation is common existed in different tumortypes. Compared with epigeneyically activated gene (DNA hypomethylation and high expression level), there are more epigenetically silenced genes (DNA hypermethylation and low expression level) across cancers.Fuetheremore, we find there are lots of genes in the calcium signaling pathway showed hypermethylation and low level of gene expression. In addition, these genes regulated by DNA methylation usually located in thekey nodes ofcalcium signaling pathways, including the control flow of sodium-calcium ion exchanger and G protein coupled receptors, etc. We can effectively distinguish the tumor samples and normal samples based on these abnormal DNA methylation sitesof calcium signaling pathway. Specially, our resultson DNA methylation in calcium signaling pathway and is consistent withRaynal’s results. Targeted DNA methylation of calcium signaling pathway genes may serverd as a newdirection for cancer therapy. Our work may be helpful for drug development research based oncalcium signaling pathways.On the other hand, we want to explore the cancer prognosis markers in gene expression level and DNA methylation level. We download gene expression and DNA methylation data of 11 solid cancer typesfrom TCGA. Compared with normal controls, weidentify differentially expressed genes in tumor, and then analyzed the correlation of gene expression and tumor stage. Finally, we identify genes with tumor prognosis which can be used as a prognostic marker acrosstumor types. In addition, we carry out GO pathway enrichment analysis in order to investigate these genes potentially involved in biological pathways. In gene expression level, results displayed genes positively related to the tumor stage aremainly enriched in cell cycle and DNA replication, including BUB1 gene in the cell cycle pathway can be servedas the prognosis of molecular tumor prognosis markers; pathways negatively related with tumor stageen riched incell adhesion and the Krebs cycle pathways. In DNA methylation level, there are some signaling pathways associated with tumor stage, including calcium signal pathway, focal adhesion, regulation of actin cytoskeleton, etc. Prognostic analysis also found that these genes related with cancer prognosis acrosstumor types.In conclusion, this study reveals that DNA hypermethylation is common existed across cancers, and DNA hypermethylation regulate genes of calcium signaling pathways and cell adhesion pathways, these genes regulated by DNA methylation often located in key nodes of calcium signaling pathways. In pan-cancer prognosis markers, we found that some genes associated with tumor stage and prognosis, these genes associated with cell cycle and DNA replication pathways, and these genes always are upregulated during the tumor stage progress, thelevel of genes expression couldbe used as a molecular marker to predict cancer survival rates. On the other hand, there are some DNA methylation sites related with tumor stage and prognosis are identified, these genes are enriched inthe calcium signaling pathways, and these of DNA methylation level can be served as predicting molecular markers of cancer survival rates. In short, our study found DNA hypermethylation in calcium signaling pathway common existed across cancer types, and DNA methylation level of the calcium signal pathway related with tumor staging and prognosis. This study is using bioinformatics methods to analyze the big data intumor research across different tumor types to explore the mechnisms of gene expression in the tumor development, it maybe useful for cancer therapy and provides sometheory support for further related cancer study, including cancer targeted therapy and precise medical. 

目录 摘要 ...................................................................................................................................................I Abstract............................................................................................................................................IV

第一章 癌症中的 DNA 甲基化及钙信号通路研究进展.................................................................1

1.1 癌症发病率及死亡人数概况........................................................................................1

1.2 癌症研究范例—癌症基因组图谱计划进展...............................................................3

1.3 癌症中 DNA 甲基化研究进展.......................................................................................5

1.3.1 表观遗传学概述...................................................................................................5

1.3.2 DNA 甲基化概述....................................................................................................7

1.3.3 DNA 甲基化与疾病的关系..................................................................................10

1.3.4 DNA 甲基化在癌症发生发展过程中的研究进展 ..............................................11

1.3.5 DNA 甲基化在癌症治疗方面的进展 ..................................................................13

1.4 癌症中的钙信号通路研究.........................................................................................15

1.4.1 钙信号通路概述..................................................................................................15

1.4.2 癌症中的钙信号通路简介.................................................................................16

1.4.3 癌症中的钙信号与癌症胞的增殖.......................................................................16

1.4.4 癌症中的钙信号与癌症转移...............................................................................17

1.4.5 癌症钙信号通路与癌症治疗...............................................................................18

1.5 本章小结......................................................................................................................18

第二章 泛肿瘤 DNA 甲基化修饰模式研究................................................................................20

2.1 研究背景.......................................................................................................................20

2.2 材料与方法..................................................................................................................21

2.3 研究结果.......................................................................................................................25

2.4 讨论..............................................................................................................................49

2.5 本章小结......................................................................................................................52

第三章 泛肿瘤预后相关 DNA 甲基化及基因表达分子标记研究...............................................53

3.1 研究背景.......................................................................................................................53

3.2 材料与方法...................................................................................................................54

3.3.结果...............................................................................................................................56

3.4 讨论..............................................................................................................................78

3.5 本章小结.....................................................................................................................79

第四章 总结与展望 ......................................................................................................................81

参考文献 ........................................................................................................................................84

作者简历及攻读学位期间发表的学术论文与研究成果...........................................................106

致谢 .....................................................................................................................................107