1、胃癌变相关分子事件研究 2、SMC4在肺腺癌中的功能及相关分子机制研究

发布时间：2018-06-17 00:42

本文选题：胃癌和癌前病变 + 胃多阶段癌变模型　；参考：《北京协和医学院》2017年博士论文

【摘要】：该博士论文由相对独立的两部分工作组成,其中第一部分分为二章。第一部分胃癌变相关分子事件研究第一章胃癌前病变及胃癌全基因组表达谱研究背景:胃癌变是一个多阶段、多因素、多基因参与的复杂过程。胃多阶段癌变模型认为正常胃粘膜进展为胃癌需要以下几个连续阶段:正常胃粘膜→非萎缩性胃炎→多灶萎缩性胃炎→肠化生→低级别上皮内瘤变→高级别上皮内瘤变→癌。利用表达谱芯片技术全面系统的对胃癌前病变及胃癌序列进行分子生物学研究,具有重要的临床和科研价值。目的:本课题旨在研究胃癌变各阶段组织在分子表达谱水平上的相似性和差异性;筛选胃癌变过程中可能起关键作用的分子,挖掘胃癌早期诊断标志物。材料和方法:本部分研究收集了 66对胃不同病变阶段新鲜组织,包括20例低级别上皮内瘤变、23例高级别上皮内瘤变、19例早期胃癌、4例进展期胃癌和配对的邻近染色正常组织,用于全基因组表达谱芯片的检测;收集了 50例胃癌患者的福尔马林浸泡石蜡包埋组织样本,一张切片上同时包括正常组织、癌前病变组织和胃癌组织,利用免疫组织化学染色(immunochemistry,IHC)对表达谱芯片数据筛选出的分子进行验证。结果:胃癌前病变及胃癌样本表达谱数据分析结果表明,在分子水平上胃癌前病变与胃癌具有相似性:很多肿瘤细胞的特征,例如DNA复制、细胞分裂、细胞周期等相关基因的上调以及生长负调控、支链氨基酸代谢等相关基因的下调,在癌前病变阶段已经显现;癌前病变组织和胃癌组织相对于邻近组织有着相似的差异表达模式,在胃癌组织中差异表达的基因绝大多在癌前病变阶段已经有了一致的差异表达。此外,胃癌前病变和胃癌在分子表达谱上存在差异性,主要涉及免疫反应和细胞粘附。一方面,我们发现562个基因可能参与了胃癌变过程,其中在蛋白互作网络中最为核心且紧密联系的8-基因模块为CD40LG、CTLA4、GZMB、ICAM1、ITGAX、SELL、TGFB1、TNF,这些分子参与了淋巴细胞和单核细胞的迁移、免疫负调控、细胞外基质组织等过程,与胃癌患者的总生存相关。另一方面,为了寻找胃癌早期诊断标志物,我们挑选了 4个在胃癌变过程中表达水平持续升高的基因进行后续免疫组化验证,结果显示,在胃癌变过程中,CCL3和CD300A的蛋白表达量和阳性率持续升高;与邻近组织相比,OSM和CHI3L1在癌前病变组织和胃癌组织中的表达量和阳性率显著升高,然而OSM和CHI3L1在癌前病变组织和胃癌组织中的表达量和阳性率没有显著差异。受试者工作特征曲线分析结果表明这四个分子对胃癌早期诊断可能具有一定的临床价值。结论:在分子水平上胃癌前病变与胃癌具有相似性,胃癌前病变可能是胃癌发生的基础。胃癌前病变和胃癌在分子表达谱上存在差异性,主要涉及免疫反应和细胞粘附,这些基因的变化可能与癌前病变向胃癌恶性转化密切相关。8-基因模块可能在胃癌变中发挥关键作用。CCL3,CD300A,CHI3L1和OSM是潜在的胃癌早期诊断标志物。以胃多阶段癌变模型作为理论基础,利用胃不同病变阶段样本对胃癌变过程进行研究,有助于阐明胃癌发生发展机制、寻找癌变关键分子和潜在的胃癌早期诊断标志物。第二章胃癌前病变及胃癌TCR免疫组库研究背景:基于肿瘤浸润淋巴细胞的免疫治疗,主要依赖于抗原和T细胞受体(T-cell receptor,TCR)的相互作用,已经在实体瘤的治疗中显现出潜在的临床应用价值。然而,TCR免疫组库的很多基本特征仍然不清楚;癌变过程中TCR免疫组库的变化,尤其是癌前病变阶段,目前还没有任何相关研究。目的:本课题旨在探究胃癌变过程中组织浸润TCR免疫组库的特征以及病变组织分子表型与病变微环境TCR免疫组库的关系。材料和方法:研究纳入了 41例不同病变阶段的胃组织样本,包括低级别上皮内瘤变、高级别上皮内瘤变、早期胃癌和配对的邻近组织。我们通过多重PCR捕获建库和IluminaHiseq2000测序平台对TCRβ互补决定区3进行了深度测序。此外,为了研究病变组织分子表型与病变微环境TCR免疫组库的关系,我们对相应的胃癌前病变样本和早期胃癌样本进行了全基因组表达谱芯片检测。结果:分析结果显示,T细胞克隆多样性和TCR免疫组库的频率分布在胃癌变过程中没有显著变化。然而,在胃癌变过程中,病变组织和配对邻近组织TCR免疫组库的相似性逐渐降低。病变组织和配对邻近组织之间的TCR免疫组库的重叠逐渐降低,为抗原驱动克隆扩增提供了直接的证据;越来越多的病变抗原相关T细胞克隆变成了高频克隆,而且它们占有的频率在胃癌变过程中逐渐增加。此外,病变组织和配对邻近组织之间的共有高频T细胞克隆的频率相似性在胃癌变不同阶段迅速降低。最后,利用基于网络的派系过滤方法对表达谱芯片数据和TCR免疫组库变异指数进行整合分析,我们找到了一个包含11个基因的模块,这11个基因的表达量与胃癌病人的总生存显著相关。结论:我们的研究阐明了在胃癌变过程中组织浸润TCR免疫组库的多阶段异质性。突变细胞和免疫系统之间的相互作用可能在细胞恶性转化过程中发挥重要作用。本研究加深了我们对胃癌变过程中免疫反应的理解。第二部分SMC4在肺腺癌中的功能及相关分子机制研究背景:越来越多的证据显示,胚胎发育和肿瘤形成在组织形态、细胞生物学学行为以及分子表达模式等方面具有极大的相似性。利用胚胎发育与肿瘤发生的相似性,重新审视正常发育的独特过程,深入挖掘胚胎发育过程中分子谱的变化规律,可以在最大程度上规避肿瘤异质性给研究带来的种种困难。染色体结构维持蛋白4(Structural maintenance of chromosome 4,SMC4)是集缩素蛋白复合体(condensin)的核心蛋白,主要参与染色体的凝集和分离。实验室前期发表的人胚胎肺发育样本和肺腺癌样本mRNA表达谱数据分析结果显示,SMC4在胚胎肺发育过程中持续下调,在肺腺癌中却呈现高表达。目的:本课题旨在探索从肺发育数据中挖掘的蛋白SMC4在肺腺癌发生发展中所发挥的功能及相关分子机制。材料和方法:首先,利用实时定量PCR(Real-timePCR)对43对手术切除的肺腺癌组织及其癌旁组织的SMC4 mRNA表达水平进行验证。接着,利用IHC对75例FFPE肺腺癌组织及其癌旁组织的SMC4蛋白表达水平进行了验证,并利用log-rank检验分析了 SMC4表达量与肺腺癌病人生存的关系。利用CCK-8实验和transwell实验检测SMC4对肺腺癌细胞增殖和侵袭的影响。为了进一步探索SMC4在肺腺癌发生发展中的机制,我们通过免疫共沉淀联合质谱分析找到了 SMC4潜在的相互作用蛋白DDX46(DEAD-boxhelicase46)。最后,利用免疫共沉淀和免疫荧光共定位实验验证SMC4和DDX46的相互作用。结果:Real-time PCR和IHC分别从RNA和蛋白水平上证实了SMC4在肺腺癌组织中的表达量显著高于癌旁组织,而且SMC4蛋白高表达是肺腺癌独立的预后不良因素(log-rank检验,p= 0.036)。细胞实验表明,SMC4敲降显著抑制A549细胞的增殖和侵袭。此外,SMC4敲降后,与增殖相关的蛋白PLK1,Cyclin B1,CDK1表达量显著下调。对人胚胎肺发育样本和肺腺癌样本mRNA表达谱数据进行分析发现,SMC4和DDX46表达模式相似;Speraman相关性分析结果表明,SMC4和DDX46的mRNA表达水平显著正相关(r=0.629,p0.0001)。正反向Co-IP证实SMC4和DDX46存在相互作用。免疫荧光共定位实验发现,SMC4和DDX46在细胞周期的各个时相均存在共定位现象。结论:SMC4在肺腺癌组织中的表达量显著高于癌旁肺组织,而且SMC4蛋白高表达是肺腺癌独立的预后不良因素。SMC4促进肺腺癌细胞的增殖和侵袭。SMC4和DDX46存在相互作用,可能通过影响pre-mRNA剪切参与肺腺癌的发生。在发育过程中与SMC4有相似表达模式的基因,可能同时参与肿瘤的形成。通过胚胎发育相关分子事件对肿瘤发生进行比较学研究,是一种挖掘潜在的肿瘤标志物和治疗靶点的有效方法,也有助于阐明肿瘤的发生发展机制。
[Abstract]:The doctoral thesis is composed of two parts, which are divided into two chapters. The first part is divided into two chapters. Part 1: the first part of the study of gastric cancer related molecular events: Chapter 1 research background of the whole genome expression profiles of gastric precancerous lesions and gastric cancer: gastric cancer is a multi stage, multi factor, complex process of multi gene participation. The normal gastric mucosa progresses to the following stages: normal gastric mucosa, non atrophic gastritis, multifocal atrophic gastritis, intestinal metaplasia, low-grade intraepithelial neoplasia, high grade intraepithelial neoplasia and cancer. It has important clinical and scientific value. Objective: the purpose of this study is to study the similarity and difference in the level of molecular expression profiles at various stages of gastric cancer, to screen the molecules that may play a key role in the process of gastric cancer change, and to excavate early diagnosis markers for gastric cancer. Materials and methods: 66 pairs of different stages of gastric lesions were collected in this study. Fresh tissue, including 20 cases of low-grade intraepithelial neoplasia, 23 cases of high grade intraepithelial neoplasia, 19 cases of early gastric cancer, 4 cases of advanced gastric cancer and paired adjacent normal tissues, were used for the detection of whole genome expression chips; 50 cases of formalin were collected and paraffin embedded tissue samples were collected in 50 cases of gastric cancer. Often tissue, precancerous tissue and gastric cancer tissue, immunochemistry (IHC) was used to verify the molecules screened by the expression spectrum chip data. Results: the analysis of the expression profiles of precancerous lesions and gastric cancer samples showed that the precancerous lesions were similar to gastric cancer at the molecular level: many tumor cells. Characteristics, such as DNA replication, cell division, cell cycle related genes up-regulated and negative regulation of growth, and downregulation of related genes, such as branched chain amino acid metabolism, have appeared in the precancerous lesion stage; precancerous tissue and gastric cancer tissues have similar differential expression patterns relative to adjacent tissues, and are differentially expressed in gastric cancer tissues. In addition, precancerous lesions and gastric cancer are different in molecular expression profiles, mainly involving immune response and cell adhesion. On the one hand, we find that 562 genes may be involved in the process of gastric cancer, the most important and closely linked in the protein interaction network. The 8- gene module of the system is CD40LG, CTLA4, GZMB, ICAM1, ITGAX, SELL, TGFB1, TNF. These molecules participate in the migration of lymphocytes and mononuclear cells, the negative regulation of immunization, the extracellular matrix, and the total survival of the gastric cancer patients. On the other hand, we choose 4 to change the process of gastric cancer in order to find the early diagnosis marker of gastric cancer. The results showed that the protein expression and positive rate of CCL3 and CD300A increased continuously during the process of gastric cancer, and the expression and positive rate of OSM and CHI3L1 in precancerous and gastric cancer tissues were significantly higher than those of adjacent tissues, while OSM and CHI3L1 were in precancerous disease. There is no significant difference in the expression and positive rate of the tissue and gastric carcinoma. The results of the work characteristic curve of the subjects show that these four molecules may have certain clinical value for the early diagnosis of gastric cancer. Conclusion: the precancerous lesions are similar to gastric cancer at the molecular level. Precancerous lesions may be the basis of gastric cancer. Precancerous lesions and gastric cancer are different in molecular expression profiles, mainly involving immune response and cell adhesion. The changes of these genes may be closely related to the malignant transformation of precancerous lesions to gastric cancer. The.8- gene module may play a key role in gastric cancer..CCL3, CD300A, CHI3L1 and OSM are potential markers for the early diagnosis of gastric cancer. The multi-stage carcinogenesis model is a theoretical basis, using samples of different stages of gastric lesions to study the process of gastric cancer. It is helpful to elucidate the mechanism of gastric cancer and development, to find the key molecules of cancer and the potential diagnostic markers for the early gastric cancer. Second the background of the study on the precancerous lesions of gastric cancer and the TCR immunization group of gastric cancer: Based on tumor infiltrating lymphocytes Immunotherapy, which is mainly dependent on the interaction of antigen and T cell receptor (T-cell receptor, TCR), has shown potential clinical value in the treatment of solid tumors. However, many basic features of the TCR immune group are still unclear, and the changes in the TCR immune group, especially the precancerous phase of the cancerous process, are not yet available. Objective: To explore the characteristics of tissue infiltrating TCR immune group during gastric cancer and the relationship between the molecular phenotype of the pathological tissue and the TCR immune group Library in the pathological microenvironment. Materials and methods: the study included 41 samples of gastric tissue in different pathological stages, including low-grade intraepithelial neoplasia and high grade intraepithelial neoplasia. Tumorigenicity, early gastric cancer and paired adjacent tissues. We sequenced the TCR beta complementation area 3 by multiple PCR capture building and IluminaHiseq2000 sequencing. In addition, in order to study the relationship between the molecular phenotype of the lesion and the TCR immune group of the pathological microenvironment, we have the corresponding precancerous lesion samples and early gastric cancer samples. The results showed that the diversity of T cell clones and the frequency distribution of the TCR immune group did not change significantly in the process of gastric cancer. However, in the process of gastric cancer, the similarity between the pathological and the paired adjacent tissues of the TCR immune group gradually decreased. The pathological tissue and the paired adjacent groups were gradually reduced. The overlap between the TCR immune group libraries of the fabric decreased gradually and provided direct evidence for the antigen driven cloning and amplification, and more and more clones of the T cells were transformed into high frequency clones, and the frequency of their occupancy increased gradually in the process of gastric cancer. In addition, the common high frequency T cells between the lesion group and the adjacent adjacent tissues were found. The frequency similarity of the clones decreased rapidly at different stages of gastric cancer. Finally, a network based factional filtering method was used to integrate the expression spectrum chip data and the TCR immune group variation index. We found a module containing 11 genes. The amount of the 11 genes was significantly related to the total survival of the gastric cancer patients. Our study elucidated the multistage heterogeneity of the tissue infiltrating TCR immune group during the process of gastric cancer. The interaction between the mutant cells and the immune system may play an important role in the process of cell malignant transformation. This study deepens our understanding of the immune response in the process of gastric cancer change. The second part of the SMC4 is in the lung adenocarcinoma. Research background: more and more evidence shows that embryonic development and tumor formation have great similarity in tissue morphology, cell biological behavior and molecular expression patterns. Using the similarity of embryo development and tumor occurrence, it reexamines the unique process of normal development and deeply excavate the embryo. The variation of molecular spectrum during fetal development can avoid the difficulties caused by tumor heterogeneity to the maximum extent. Chromosome structure maintenance protein 4 (Structural maintenance of chromosome 4, SMC4) is the core egg white of the aggregation protein complex (condensin), which is mainly involved in the agglutination and separation of chromosomes. The results of human embryonic lung development samples and mRNA expression profiles of lung adenocarcinoma samples showed that SMC4 was down regulated during embryonic lung development and showed high expression in lung adenocarcinoma. Objective: the purpose of this study is to explore the function and related molecules of protein SMC4 in the development and development of lung adenocarcinoma from lung development data. Mechanism, materials and methods: first, using real-time quantitative PCR (Real-timePCR) to verify the expression level of SMC4 mRNA in 43 pairs of surgically resected lung adenocarcinoma tissues and adjacent tissues. Then, the expression of SMC4 protein in 75 cases of FFPE lung adenocarcinoma tissue and its para cancerous tissues was verified by IHC, and SMC4 was analyzed by log-rank test. The relationship between expression and survival of lung adenocarcinoma patients. The effects of SMC4 on proliferation and invasion of lung adenocarcinoma cells were detected by CCK-8 and Transwell experiments. In order to further explore the mechanism of SMC4 in the development of lung adenocarcinoma, we found the potential interaction protein DDX46 (DEAD-boxhelica) by immunoprecipitation combined with mass spectrometry analysis. Se46). Finally, the interaction between immunofluorescence and immunofluorescence was used to verify the interaction between SMC4 and DDX46. Results: Real-time PCR and IHC showed that the expression of SMC4 in lung adenocarcinoma tissues was significantly higher than that in the para cancerous tissues from RNA and protein levels, and the high expression of SMC4 protein was an independent prognostic factor for lung adenocarcinoma (log-rank). Test, p= 0.036). Cell experiments showed that SMC4 knockdown significantly inhibited the proliferation and invasion of A549 cells. In addition, after SMC4 knockdown, the expression of PLK1, Cyclin B1, CDK1 was significantly down after the SMC4 knockdown. The SMC4 and DDX46 expression patterns were found to be similar to the SMC4 and DDX46 expression patterns for the human fetal lung development samples and lung adenocarcinoma samples; The results of the correlation analysis showed that the mRNA expression level of SMC4 and DDX46 was positively correlated (r=0.629, P0.0001). The positive and reverse Co-IP confirmed the interaction between SMC4 and DDX46. The co localization of SMC4 and DDX46 in each phase of the cell cycle was found in the immunofluorescence co localization experiment. Conclusion: SMC4 in the lung adenocarcinoma tissues was significantly higher than that of SMC4. Para cancerous lung tissue, and high expression of SMC4 protein is an independent prognostic factor for lung adenocarcinoma,.SMC4 promotes the proliferation of lung adenocarcinoma cells and the interaction of.SMC4 and DDX46, and may be involved in the occurrence of lung adenocarcinoma by affecting pre-mRNA shear. In the process of development, the gene similar to SMC4 may be involved in the shape of the tumor at the same time. The comparative study of tumorigenesis through embryonic development related molecular events is an effective method for mining potential tumor markers and targets, and also helps to elucidate the mechanism of tumor development and development.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.2

【相似文献】