人源性胃癌小鼠皮下移植瘤模型建立及传代过程中的遗传忠实性研究

发布时间：2018-03-14 09:29

  本文选题：胃癌　切入点：基因组　出处：《武汉大学》2016年博士论文　论文类型：学位论文

【摘要】：背景：随着精准医疗时代的到来,临床前研究成为了不可或缺的阶段,而胃癌动物模型的建立不仅为临床前研究提供了实验可行性基础,也在探索胃癌的发生机制、确立临床诊疗方案等方面发挥着重要的作用。然而,胃癌PDXs小鼠皮下移植瘤模型与患者肿瘤的相似度到底多大、在传代过程中又发生着怎样的动态变化并不清楚,这从很大程度上影响着利用临床前试验检测药效并向临床转化过程的可靠性。尤其随着DNA甲基化在癌症中的重要作用不断被发现,基于表观遗传学的潜在药物也不断涌现。在这样的背景下,从多组学角度研究胃癌PDXs小鼠皮下移植瘤模型建立以及传代移植过程中的变化就显得尤为重要、紧迫。目的：旨在揭示胃癌PDXS小鼠皮下移植瘤模型建立以及传代移植过程中基因组、转录组及DNA甲基化组的动态变化规律和遗传学忠实性情况,为探讨运用胃癌PDXs小鼠皮下移植瘤模型在临床前试验检测药效、并向临床转化过程中的可靠性提供重要依据。方法：首先建立胃癌PDXs小鼠皮下移植瘤模型,并以该传代模型和相应的原癌组织为对象,借助于Illumina高通量测序平台对目标样本进行基因组、转录组及DNA甲基化组的测序,继而以UCSC hg19为参考基因组、运用基因组学、转录组、生物信息学及系统生物学的理论与方法,从组学水平对序列变异、基因表达及DNA甲基化水平进行鉴定；然后从两个层次(分别为：各代胃癌PDXs小鼠皮下移植瘤模型与原癌组织之间的比较以及胃癌PDXs小鼠皮下移植瘤模型的各个传代之间的比较)对所获的数据进行比较分析,为系统揭示胃癌PDXs小鼠模型与原癌组织间的遗传忠实性提供数据基础；然后通过收集FDA认证的癌症药物及靶标基因信息,并从多组学分析癌症药物靶标基因在胃癌PDXs小鼠皮下移植瘤模型建立以及传代过程中的动态变化。结果：通过组学分析,各代移植瘤与原癌组织的遗传相似度均约95%；进而对差异部分进行分析。首先,对各代之间的生物学样本进行了基因组水平的变异分析,发现原癌组织中未发生突变、仅在移植瘤中发现突变的基因有35个,功能富集分析显示这些基因主要与GO:0060759 regulation of response to cytokine stimulus、GO:0008202 steroid metabolic process以及GO:0005244 voltage-gated ion channel activity相关；其次,通过转录组水平的表达分析,发现与原癌组织相比、在传代移植瘤中均显著差异表达(qvalue0.05)的基因有337个(上调基因12个和下调325个),这些基因主要参与的信号通路包括Focal adhesion、Extracellular matrix organization以及Inflammation mediated by chemokine and cytokine signaling pathway;对lncRNA的表达分析发现有10个lncRN在各代移植瘤中均显著差异表达,其中仅RP11-72L22.1基因显著上调,其他均显著下调；再次,根据所测得DNA甲基化组数据,发现移植瘤的甲基化水平均高于原癌组织,传代移植瘤中共同的DMR区域43个,其中有26个高甲基化区域,17个低甲基化区域；对显著差异的DNA甲基化区域的基因进行pathway富集分析发现,传代移植瘤中有3个共同的显著富集pathway与心肌病相关：Arrhythmogenic right ventricular cardiomyopathy (ARVC)、Hypertrophic cardiomyopathy (HCM)和Dilated cardiomyopathy (DCM),显示胃癌PDXs小鼠皮下移植瘤与心肌病之间的潜在联系；最后,通过收集FDA认证的癌症药物及靶标基因,并从多组学分析靶标基因在胃癌PDXs小鼠皮下移植瘤模型建立以及传代过程中的动态变化,发现5个药靶基因在移植瘤中已发生突变或基因融合事件；此外,传代移植瘤中有5个共同胃癌药靶基因出现显著差异表达(PGFRA_HUMAN、PGFRB_HUMAN、VGFR1_HUMAN、VGFR2_HUMAN 和DDR2_HUMAN),针对这些靶标的胃癌药物为Imatinib Mesylate、Sunitinib Malate、Ramucirumab和Regorafenib,且这些基因共同参与的pathway有2个,即Cytokine-cytokine receptor interaction 和 Focal adhesion信号通路；甲基化组分析发现22个共同的药靶基因在差异甲基化区域,其中仅1个是FDA认证的胃癌药物的靶标(ABL1_HUMAN)；表明与这些靶基因相关的靶向药物的临床前药效验证将可能受到影响。结论：(1)原癌组织在传代的各代动物模型中都能保持一定的遗传学忠实性,相对稳定的遗传可为我们设计胃癌靶标药物提供依据；(2)基因组水平的变异分析和对转录组水平的表达分析,发现原癌组织中未发生突变、仅在移植瘤中发现突变的基因有35个,在传代移植瘤中均显著差异表达的基因有337个(上调基因12个和下调325个),功能分析显示这些基因变异主要与肿瘤组织适应新的微环境相关；(3)根据对所测得DNA甲基化组数据,发现移植瘤的甲基化水平均高于原癌组织,传代移植瘤中共同的DMR区域43个,其中有26个高甲基化区域,17个低甲基化区域；功能分析显示主要与特定的癌症信号通路相关；(4)通过收集FDA认证的癌症药物及靶标基因,首次发现5个药靶基因在移植瘤中已发生突变或基因融合事件；转录组分析显示传代移植瘤中有5个胃癌药靶基因发生表达显著差异；甲基化组分析发现22个共同的药靶基因在差异甲基化区域,其中仅1个是FDA认证的胃癌药物的靶标(ABL1_HUMAN);因此在临床应用针对这些靶标的胃癌药物时应该注意其对治疗效果的影响。
[Abstract]:Background: with the precise medical era, preclinical research has become an important stage, and the establishment of animal model of gastric cancer is not only a pre clinical study provides an experimental basis of feasibility, is also exploring the pathogenesis of gastric cancer, plays an important role in the establishment of clinical diagnosis and treatment. However, in the end how much similarity between the mouse gastric cancer PDXs subcutaneous transplantation tumor model and tumor patients, during passage and changing what is not clear, it has great influence on the use of pre clinical testing and clinical efficacy to the reliability of the transformation process. Especially with the important role of DNA methylation in cancer have been discovered, potential epigenetic drugs are constantly emerging. Based on this background, from the perspective of multiple groups of model mouse gastric cancer PDXs subcutaneous tumor established and serial transplantation process The change is particularly important and urgent. Objective: to reveal the genomic model of gastric cancer PDXS mice subcutaneous transplantation tumor and establish serial transplantation process, dynamic changes and genetic transcription Xuezhong solid methylation group and DNA group, to explore the use of gastric cancer in PDXs mice skin transplantation tumor model down in preclinical testing effect it provides an important basis to the clinical reliability in the process of transformation. Methods: firstly, establish the model of gastric cancer in PDXs mice subcutaneously transplanted tumor, and with the passage model and the corresponding original cancer as the object, with the help of Illumina high-throughput sequencing platform for the genome of target samples, sequencing transcriptome and DNA methylation group, followed by UCSC the use of hg19 as a reference genome, genomics, transcriptomics, theories and methods of bioinformatics and systems biology, from the group level of sequence variation, gene expression and DNA methylation Identify the level; then from two levels (respectively: a comparison between the model of the generation of gastric cancer PDXs mice subcutaneous transplantation tumor and primary carcinoma and gastric cancer PDXs mouse subcutaneous transplantation tumor model of each passage) to make a comparative analysis of the data, provide the data base for the system to reveal the genetic fidelity gastric cancer PDXs mice model and original carcinoma; then through cancer drugs and target gene information collection of FDA certification, and from the proteomic analysis of cancer drug target gene in gastric cancer model in PDXs mice subcutaneously transplanted tumor established and dynamic change of generations in the process. Results: through proteomic analysis, the genetic similarity of the transplanted tumor and primary tumor tissues were about 95%; and then to difference parts. Firstly, the biological samples each generation were analyzed between the variation of genome level, primary carcinoma No mutations were found only in tumor mutations in 35 genes, enrichment analysis showed that these genes were mainly associated with GO:0060759 regulation of response to cytokine stimulus, GO:0008202 steroid metabolic process GO:0005244 voltage-gated ion channel and activity; secondly, through the expression of transcriptome analysis, found that compared with the original cancer tissue, the expression of there were significant differences in the passage in transplanted tumor (qvalue0.05) and 337 genes (12 up-regulated genes and 325 down regulated), these genes are mainly involved in the signaling pathway including Focal adhesion, Extracellular matrix and organization Inflammation mediated by chemokine and cytokine signaling pathway; the expression of lncRNA analysis found that the expression of 10 lncRN significant differences in the transplanted tumor, which only RP11-72L22.1 gene was up-regulated significantly in the other Reduced; again, based on the measured data of DNA methylation group, found that methylation of transplanted tumor was higher than the original average cancer xenografts in DMR region, were common in 43, including 26 high methylation, 17 low methylation; pathway enrichment analysis found that the methylation of DNA region significant differences in the gene, were transplanted tumor in 3 common significant enrichment of pathway and Arrhythmogenic: right ventricular cardiomyopathy related cardiomyopathy (ARVC), Hypertrophic cardiomyopathy (HCM) and Dilated cardiomyopathy (DCM), shows the potential link between tumor and cardiomyopathy mouse gastric cancer PDXs subcutaneous transplantation; finally, the cancer drug and target gene collect the FDA certification, and from the proteomic analysis of dynamic changes of target genes in gastric cancer model established PDXs mice subcutaneous transplantation tumor and the passage in the process, found the 5 drug target By mutation or gene fusion events have occurred in the transplanted tumor; in addition, the passage in transplanted tumor appeared significant differences in expression of the 5 common cancer drug target genes (PGFRA_HUMAN, PGFRB_HUMAN, VGFR1_HUMAN, VGFR2_HUMAN and DDR2_HUMAN), for these targets for gastric cancer Imatinib Mesylate, Sunitinib Malate, Ramucirumab and Regorafenib, and these genes together in pathway 2, Cytokine-cytokine receptor interaction and Focal adhesion signaling pathway; methylation group analysis showed that 22 common drug target genes in differentially methylated regions, of which only 1 are gastric cancer FDA approved drug target (ABL1_HUMAN); that associated with these target gene targeting preclinical pharmacodynamics validation the drugs will be affected. Conclusion: (1) primary carcinoma can maintain the genetic fidelity in each generation of animal models in the passage, phase We can provide the basis for the design of gastric cancer drug target genetic stable; (2) analysis of genome-wide variation analysis and transcriptome expression, found no mutation in primary cancer tissues, only found in tumor mutations in 35 genes, there were significant differences in the expression of genes in tumor passage there are 337 (12 up-regulated genes and 325 down regulated), functional analysis revealed that this gene mutation and tumor tissues adapt to new micro environment; (3) according to the measured data of DNA methylation group, found that the methylation level of transplanted tumor were higher than primary carcinoma, were transplanted in DMR the common area of 43, including 26 high methylation, 17 low methylation analysis showed that the main functional areas; and cancer specific signaling pathways related; (4) the cancer drug target gene and collection of FDA certification, for the first time found that the 5 drug target base By mutation or gene fusion events have occurred in the transplanted tumor; transcriptome analysis showed that the passage of transplanted tumor in 5 gastric cancer drug target gene expression difference; methylation group analysis found 22 common drug target genes in differentially methylated regions, of which only 1 are gastric cancer FDA approved drug targets (ABL1_HUMAN); so in the clinical application of gastric cancer drugs against these targets should pay attention to its impact on the treatment effect.

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.2;R-332
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本文编号：1610610