胃粘膜上皮癌变相关蛋白质分子的筛选与鉴定

发布时间：2018-09-09 13:19

【摘要】：目的:胃癌是我国及亚太地区最常见的恶性肿瘤之一。本研究旨在寻找新的胃癌诊断蛋白质分子,进一步阐明胃粘膜上皮癌变的分子机制。方法:1.采用激光捕获显微切割(LCM)技术,分别获取高纯度的正常胃粘膜(NGM)、非典型增生(AH)、低分化腺癌(GPDAC)及淋巴结转移癌(LMGAC)组织,采用同位素标记相对和绝对定量(iTRAQ)结合二维液相色谱联用质谱(2D LC-MS/MS)技术,寻找胃粘膜上皮癌变过程中不同阶段的差异表达蛋白质分子。2.选择PRSS1、HSP90α/β、TGM2、SerpinA3和P180蛋白质进行进一步的验证与分析。首先采用Western-blot,检测5个蛋白质在NGM、AH、GPDAC及LMGAC中的表达情况;其次通过免疫组织化学染色,观察5个蛋白质在组织芯片(包含NGM、AH、GAC及LMGAC)中的表达情况,检测结果采用ROC曲线分析,揭示5个蛋白质表达对胃腺癌早期诊断的评估作用。3.构建pcDNA3.1-PRSS1真核表达载体,转染GES-1细胞,建立了PRSS1高表达的GES-1细胞系(GES-1/pcDNA3.1-PRSS1),同时构建pcDNA6.2-GW/EmGFP-miR-PRSS1干扰载体,转染MGC803细胞,建立PRSS1低表达的MGC803(MGC803/pcDNA6.2-GW/EmGFP-miR-PRSS1)细胞系;通过绘制细胞生长曲线、平皿克隆形成实验、软琼脂集落形成实验、流式细胞术、Hoechest33258染色、细胞划痕迁移实验、Transwell迁移与侵袭实验,观察PRSS1表达改变对GES-1和MGC803细胞增殖、周期、凋亡、迁移与侵袭的影响;最后采用Western-blot检测PRSS1表达改变对GES-1和mgc803细胞中wnt1/β-catenin信号通路的影响。结果:1.本研究总共鉴定出胃粘膜上皮癌变相关差异表达蛋白质243个,在胃癌中表达上调的蛋白质153个,下调者90个,有的蛋白质在ngm、ah、gpdac及lmgac中表达均上调,有的则均下调,有的呈阶段性改变。通过分层聚类分析,将243个差异表达蛋白质聚集成3大群与8个簇。go功能注释显示,第1大群蛋白质的功能主要与细胞生长、凋亡、翻译与代谢等有关;第2大群蛋白质主要与器官发育、细胞凋亡与生物刺激应答等有关;第3大群蛋白质主要与细胞增殖、凋亡、代谢、转运、分子定位与免疫应答等有关。通过kegg通路分析,三大群蛋白质中参与一些肿瘤相关的信号通路,如细胞周期与凋亡、mapk、p53及erbb信号通路等。2.联合检测prss1、hsp90α/β、tgm2、serpina3和p180蛋白质表达,判别ngm与ah的敏感性和特异性分别为88%和84%,判别ngm与gac的敏感性和特异性分别为86%和85%,判别ngm与lmgac的敏感性和特异性分别为89%和87%,判别ah与gac的敏感性和特异性分别为88%和84%,判别ah与lmgac的敏感性和特异性分别为87%和83%。结果表明,prss1、hsp90α/β、tgm2、serpina3和p180联合检测,可用于正常胃粘膜、非典型增生、胃腺癌和淋巴结转移癌的鉴别。3.ges-1/pcdna3.1-prss1较ges-1/pcdna3.1(对照细胞)和未转染的ges-1细胞生长速度加快、平皿克隆和软琼脂集落数均增加、细胞凋亡率降低、细胞迁移与侵袭能力增强;mgc803/pcdna6.2-gw/emgfp-mir-prss1较mgc803/pcdna6.2-gw/emgfp-mir(对照细胞)和未转染的mgc803细胞生长速度降低、平皿克隆和软琼脂集落数均减少、细胞凋亡率增加、细胞迁移与侵袭能力减弱;PRSS1高表达促使GSE-1细胞中Wnt1与β-catenin蛋白质表达增加,而β-catenin蛋白磷酸化水平降低,PRSS1低表达使MGC803细胞中Wnt1与β-catenin蛋白质表达降低,而β-catenin蛋白磷酸化水平升高;PRSS1高表达促使GES-1细胞生长与增殖、迁移与侵袭能力增强,细胞增殖指数增高,细胞凋亡率下降,Wnt1/β-catenin信号通路活化;而PRSS1低表达则使MGC803细胞生长与增殖、迁移与侵袭能力降低,细胞增殖指数下降,细胞凋亡率减少,Wnt1/β-catenin信号通路抑制。结论:1.胃粘膜上皮癌变过程中发生了蛋白质表达谱的改变,本研究鉴定出相关蛋白质243个。2.联合检测PRSS1、HSP90α/β、TGM2、SerpinA3和P180蛋白质表达,可为胃癌早期诊断提供参考。3.PRSS1通过影响Wnt1/β-catenin信号通路,调节细胞的生长与增殖、迁移及侵袭。
[Abstract]:Objective: Gastric cancer is one of the most common malignant tumors in China and Asia-Pacific region. The aim of this study is to find a new protein molecule for the diagnosis of gastric cancer and further elucidate the molecular mechanism of gastric epithelial carcinogenesis. Differentially expressed proteins in different stages of gastric mucosal epithelial carcinogenesis were identified by using relative and absolute quantitative isotope labeling (iTRAQ) and two-dimensional liquid chromatography-mass spectrometry (2D LC-MS/MS) techniques. 2. PRSS1, HSP90 alpha/beta, TGM2, SerpinA3 and P180 proteins were selected to enter gastric mucosal epithelial carcinogenesis. Firstly, Western-blot was used to detect the expression of five proteins in NGM, AH, GPDAC and LMGAC, and then immunohistochemical staining was used to observe the expression of five proteins in tissue microarray (including NGM, AH, GAC and LMGAC). ROC curve analysis was used to reveal the expression of five proteins in gastric adenocarcinoma. Establishment of pcDNA3.1-PRSS1 eukaryotic expression vector, transfection of GES-1 cells, establishment of PRSS1 overexpression GES-1 cell line (GES-1/pcDNA3.1-PRSS1), and construction of pcDNA6.2-GW/EmGFP-microRNA-PRSS1 interference vector, transfection of MGC803 cells, low PRSS1 expression MGC803 (MGC803/pcDNA6.2-GW/EmGFP-microRNA-PRSS1) cell line; The effects of PRSS1 expression on proliferation, cycle, apoptosis, migration and invasion of GES-1 and MGC803 cells were observed by plotting cell growth curve, plate cloning, soft agar colony formation, flow cytometry, Hoechest 33258 staining, scratch migration, and Transwell migration and invasion experiments. Results: 1. A total of 243 differentially expressed proteins associated with gastric carcinogenesis were identified in GES-1 and MGC803 cells. 153 proteins were up-regulated and 90 were down-regulated in gastric cancer. Some of them were up-regulated in ngm, ah, gpdac and lmgac, while others were down-regulated. By hierarchical cluster analysis, 243 differentially expressed proteins were clustered into 3 groups and 8 clusters.go functional annotations showed that the functions of the first group of proteins were mainly related to cell growth, apoptosis, translation and metabolism. Large groups of proteins are mainly related to cell proliferation, apoptosis, metabolism, transport, molecular localization and immune response. The sensitivity and specificity of NGM and ah were 88% and 84%, 86% and 85% respectively. The sensitivity and specificity of NGM and lmgac were 89% and 87%, 88% and 84% respectively. The sensitivity and specificity of ah and lmgac were 87% and 83% respectively. The results showed that prss1, Hsp90 alpha / beta, TGM 2, serpina 3 and P180 could be used to differentiate normal gastric mucosa, atypical hyperplasia, gastric adenocarcinoma and lymph node metastasis. 3. GES-1 / pcDNA 3.1-prss1 cells grew faster than GES-1 / pcDNA 3.1 (control cells) and untransfected GES-1 cells, the number of plate clones and soft agar colony increased, and the apoptosis rate increased. Compared with MGC803 / pcDNA 6.2-gw / emgfp-mir-prss1 and MGC803 / pcDNA 6.2-gw / emgfp-mir (control cells) and mgc803, the growth rate of MGC803 Cells was decreased, the number of colonies of plate clones and soft agar was decreased, the apoptosis rate was increased, and the migration and invasion ability was weakened. The expression of Wnt1 and beta-catenin protein increased, while the phosphorylation level of beta-catenin protein decreased. The low expression of PRSS1 decreased the expression of Wnt1 and beta-catenin protein, while the phosphorylation level of beta-catenin protein increased in MGC803 cells. The apoptosis rate decreased and the Wnt1/beta-catenin signaling pathway was activated, while the low expression of PRSS1 decreased the growth and proliferation, migration and invasion ability, cell proliferation index, apoptosis rate and Wnt1/beta-catenin signaling pathway of MGC803 cells. The expression of PRSS1, HSP90 alpha/beta, TGM2, SerpinA3 and P180 proteins was detected. PRSS1 could regulate cell growth, proliferation, migration and invasion by affecting Wnt1/beta-catenin signaling pathway.
【学位授予单位】：南华大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.2

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