本文選題：胃癌　切入點：基因組　出處：《武漢大學》2016年博士論文　論文類型：學位論文

【摘要】：背景：隨著精準醫(yī)療時代的到來,臨床前研究成為了不可或缺的階段,而胃癌動物模型的建立不僅為臨床前研究提供了實驗可行性基礎(chǔ),也在探索胃癌的發(fā)生機制、確立臨床診療方案等方面發(fā)揮著重要的作用。然而,胃癌PDXs小鼠皮下移植瘤模型與患者腫瘤的相似度到底多大、在傳代過程中又發(fā)生著怎樣的動態(tài)變化并不清楚,這從很大程度上影響著利用臨床前試驗檢測藥效并向臨床轉(zhuǎn)化過程的可靠性。尤其隨著DNA甲基化在癌癥中的重要作用不斷被發(fā)現(xiàn),基于表觀遺傳學的潛在藥物也不斷涌現(xiàn)。在這樣的背景下,從多組學角度研究胃癌PDXs小鼠皮下移植瘤模型建立以及傳代移植過程中的變化就顯得尤為重要、緊迫。目的：旨在揭示胃癌PDXS小鼠皮下移植瘤模型建立以及傳代移植過程中基因組、轉(zhuǎn)錄組及DNA甲基化組的動態(tài)變化規(guī)律和遺傳學忠實性情況,為探討運用胃癌PDXs小鼠皮下移植瘤模型在臨床前試驗檢測藥效、并向臨床轉(zhuǎn)化過程中的可靠性提供重要依據(jù)。方法：首先建立胃癌PDXs小鼠皮下移植瘤模型,并以該傳代模型和相應的原癌組織為對象,借助于Illumina高通量測序平臺對目標樣本進行基因組、轉(zhuǎn)錄組及DNA甲基化組的測序,繼而以UCSC hg19為參考基因組、運用基因組學、轉(zhuǎn)錄組、生物信息學及系統(tǒng)生物學的理論與方法,從組學水平對序列變異、基因表達及DNA甲基化水平進行鑒定；然后從兩個層次(分別為：各代胃癌PDXs小鼠皮下移植瘤模型與原癌組織之間的比較以及胃癌PDXs小鼠皮下移植瘤模型的各個傳代之間的比較)對所獲的數(shù)據(jù)進行比較分析,為系統(tǒng)揭示胃癌PDXs小鼠模型與原癌組織間的遺傳忠實性提供數(shù)據(jù)基礎(chǔ)；然后通過收集FDA認證的癌癥藥物及靶標基因信息,并從多組學分析癌癥藥物靶標基因在胃癌PDXs小鼠皮下移植瘤模型建立以及傳代過程中的動態(tài)變化。結(jié)果：通過組學分析,各代移植瘤與原癌組織的遺傳相似度均約95%；進而對差異部分進行分析。首先,對各代之間的生物學樣本進行了基因組水平的變異分析,發(fā)現(xiàn)原癌組織中未發(fā)生突變、僅在移植瘤中發(fā)現(xiàn)突變的基因有35個,功能富集分析顯示這些基因主要與GO:0060759 regulation of response to cytokine stimulus、GO:0008202 steroid metabolic process以及GO:0005244 voltage-gated ion channel activity相關(guān)；其次,通過轉(zhuǎn)錄組水平的表達分析,發(fā)現(xiàn)與原癌組織相比、在傳代移植瘤中均顯著差異表達(qvalue0.05)的基因有337個(上調(diào)基因12個和下調(diào)325個),這些基因主要參與的信號通路包括Focal adhesion、Extracellular matrix organization以及Inflammation mediated by chemokine and cytokine signaling pathway;對lncRNA的表達分析發(fā)現(xiàn)有10個lncRN在各代移植瘤中均顯著差異表達,其中僅RP11-72L22.1基因顯著上調(diào),其他均顯著下調(diào)；再次,根據(jù)所測得DNA甲基化組數(shù)據(jù),發(fā)現(xiàn)移植瘤的甲基化水平均高于原癌組織,傳代移植瘤中共同的DMR區(qū)域43個,其中有26個高甲基化區(qū)域,17個低甲基化區(qū)域；對顯著差異的DNA甲基化區(qū)域的基因進行pathway富集分析發(fā)現(xiàn),傳代移植瘤中有3個共同的顯著富集pathway與心肌病相關(guān)：Arrhythmogenic right ventricular cardiomyopathy (ARVC)、Hypertrophic cardiomyopathy (HCM)和Dilated cardiomyopathy (DCM),顯示胃癌PDXs小鼠皮下移植瘤與心肌病之間的潛在聯(lián)系；最后,通過收集FDA認證的癌癥藥物及靶標基因,并從多組學分析靶標基因在胃癌PDXs小鼠皮下移植瘤模型建立以及傳代過程中的動態(tài)變化,發(fā)現(xiàn)5個藥靶基因在移植瘤中已發(fā)生突變或基因融合事件；此外,傳代移植瘤中有5個共同胃癌藥靶基因出現(xiàn)顯著差異表達(PGFRA_HUMAN、PGFRB_HUMAN、VGFR1_HUMAN、VGFR2_HUMAN 和DDR2_HUMAN),針對這些靶標的胃癌藥物為Imatinib Mesylate、Sunitinib Malate、Ramucirumab和Regorafenib,且這些基因共同參與的pathway有2個,即Cytokine-cytokine receptor interaction 和 Focal adhesion信號通路；甲基化組分析發(fā)現(xiàn)22個共同的藥靶基因在差異甲基化區(qū)域,其中僅1個是FDA認證的胃癌藥物的靶標(ABL1_HUMAN)；表明與這些靶基因相關(guān)的靶向藥物的臨床前藥效驗證將可能受到影響。結(jié)論：(1)原癌組織在傳代的各代動物模型中都能保持一定的遺傳學忠實性,相對穩(wěn)定的遺傳可為我們設計胃癌靶標藥物提供依據(jù)；(2)基因組水平的變異分析和對轉(zhuǎn)錄組水平的表達分析,發(fā)現(xiàn)原癌組織中未發(fā)生突變、僅在移植瘤中發(fā)現(xiàn)突變的基因有35個,在傳代移植瘤中均顯著差異表達的基因有337個(上調(diào)基因12個和下調(diào)325個),功能分析顯示這些基因變異主要與腫瘤組織適應新的微環(huán)境相關(guān)；(3)根據(jù)對所測得DNA甲基化組數(shù)據(jù),發(fā)現(xiàn)移植瘤的甲基化水平均高于原癌組織,傳代移植瘤中共同的DMR區(qū)域43個,其中有26個高甲基化區(qū)域,17個低甲基化區(qū)域；功能分析顯示主要與特定的癌癥信號通路相關(guān)；(4)通過收集FDA認證的癌癥藥物及靶標基因,首次發(fā)現(xiàn)5個藥靶基因在移植瘤中已發(fā)生突變或基因融合事件；轉(zhuǎn)錄組分析顯示傳代移植瘤中有5個胃癌藥靶基因發(fā)生表達顯著差異；甲基化組分析發(fā)現(xiàn)22個共同的藥靶基因在差異甲基化區(qū)域,其中僅1個是FDA認證的胃癌藥物的靶標(ABL1_HUMAN);因此在臨床應用針對這些靶標的胃癌藥物時應該注意其對治療效果的影響。
[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.

【學位授予單位】：武漢大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R735.2;R-332
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本文編號：1610610