胃粘膜上皮癌變相關(guān)蛋白質(zhì)分子的篩選與鑒定
發(fā)布時間：2018-09-09 13:19
【摘要】：目的:胃癌是我國及亞太地區(qū)最常見的惡性腫瘤之一。本研究旨在尋找新的胃癌診斷蛋白質(zhì)分子,進一步闡明胃粘膜上皮癌變的分子機制。方法:1.采用激光捕獲顯微切割(LCM)技術(shù),分別獲取高純度的正常胃粘膜(NGM)、非典型增生(AH)、低分化腺癌(GPDAC)及淋巴結(jié)轉(zhuǎn)移癌(LMGAC)組織,采用同位素標(biāo)記相對和絕對定量(iTRAQ)結(jié)合二維液相色譜聯(lián)用質(zhì)譜(2D LC-MS/MS)技術(shù),尋找胃粘膜上皮癌變過程中不同階段的差異表達蛋白質(zhì)分子。2.選擇PRSS1、HSP90α/β、TGM2、SerpinA3和P180蛋白質(zhì)進行進一步的驗證與分析。首先采用Western-blot,檢測5個蛋白質(zhì)在NGM、AH、GPDAC及LMGAC中的表達情況;其次通過免疫組織化學(xué)染色,觀察5個蛋白質(zhì)在組織芯片(包含NGM、AH、GAC及LMGAC)中的表達情況,檢測結(jié)果采用ROC曲線分析,揭示5個蛋白質(zhì)表達對胃腺癌早期診斷的評估作用。3.構(gòu)建pcDNA3.1-PRSS1真核表達載體,轉(zhuǎn)染GES-1細(xì)胞,建立了PRSS1高表達的GES-1細(xì)胞系(GES-1/pcDNA3.1-PRSS1),同時構(gòu)建pcDNA6.2-GW/EmGFP-miR-PRSS1干擾載體,轉(zhuǎn)染MGC803細(xì)胞,建立PRSS1低表達的MGC803(MGC803/pcDNA6.2-GW/EmGFP-miR-PRSS1)細(xì)胞系;通過繪制細(xì)胞生長曲線、平皿克隆形成實驗、軟瓊脂集落形成實驗、流式細(xì)胞術(shù)、Hoechest33258染色、細(xì)胞劃痕遷移實驗、Transwell遷移與侵襲實驗,觀察PRSS1表達改變對GES-1和MGC803細(xì)胞增殖、周期、凋亡、遷移與侵襲的影響;最后采用Western-blot檢測PRSS1表達改變對GES-1和mgc803細(xì)胞中wnt1/β-catenin信號通路的影響。結(jié)果:1.本研究總共鑒定出胃粘膜上皮癌變相關(guān)差異表達蛋白質(zhì)243個,在胃癌中表達上調(diào)的蛋白質(zhì)153個,下調(diào)者90個,有的蛋白質(zhì)在ngm、ah、gpdac及l(fā)mgac中表達均上調(diào),有的則均下調(diào),有的呈階段性改變。通過分層聚類分析,將243個差異表達蛋白質(zhì)聚集成3大群與8個簇。go功能注釋顯示,第1大群蛋白質(zhì)的功能主要與細(xì)胞生長、凋亡、翻譯與代謝等有關(guān);第2大群蛋白質(zhì)主要與器官發(fā)育、細(xì)胞凋亡與生物刺激應(yīng)答等有關(guān);第3大群蛋白質(zhì)主要與細(xì)胞增殖、凋亡、代謝、轉(zhuǎn)運、分子定位與免疫應(yīng)答等有關(guān)。通過kegg通路分析,三大群蛋白質(zhì)中參與一些腫瘤相關(guān)的信號通路,如細(xì)胞周期與凋亡、mapk、p53及erbb信號通路等。2.聯(lián)合檢測prss1、hsp90α/β、tgm2、serpina3和p180蛋白質(zhì)表達,判別ngm與ah的敏感性和特異性分別為88%和84%,判別ngm與gac的敏感性和特異性分別為86%和85%,判別ngm與lmgac的敏感性和特異性分別為89%和87%,判別ah與gac的敏感性和特異性分別為88%和84%,判別ah與lmgac的敏感性和特異性分別為87%和83%。結(jié)果表明,prss1、hsp90α/β、tgm2、serpina3和p180聯(lián)合檢測,可用于正常胃粘膜、非典型增生、胃腺癌和淋巴結(jié)轉(zhuǎn)移癌的鑒別。3.ges-1/pcdna3.1-prss1較ges-1/pcdna3.1(對照細(xì)胞)和未轉(zhuǎn)染的ges-1細(xì)胞生長速度加快、平皿克隆和軟瓊脂集落數(shù)均增加、細(xì)胞凋亡率降低、細(xì)胞遷移與侵襲能力增強;mgc803/pcdna6.2-gw/emgfp-mir-prss1較mgc803/pcdna6.2-gw/emgfp-mir(對照細(xì)胞)和未轉(zhuǎn)染的mgc803細(xì)胞生長速度降低、平皿克隆和軟瓊脂集落數(shù)均減少、細(xì)胞凋亡率增加、細(xì)胞遷移與侵襲能力減弱;PRSS1高表達促使GSE-1細(xì)胞中Wnt1與β-catenin蛋白質(zhì)表達增加,而β-catenin蛋白磷酸化水平降低,PRSS1低表達使MGC803細(xì)胞中Wnt1與β-catenin蛋白質(zhì)表達降低,而β-catenin蛋白磷酸化水平升高;PRSS1高表達促使GES-1細(xì)胞生長與增殖、遷移與侵襲能力增強,細(xì)胞增殖指數(shù)增高,細(xì)胞凋亡率下降,Wnt1/β-catenin信號通路活化;而PRSS1低表達則使MGC803細(xì)胞生長與增殖、遷移與侵襲能力降低,細(xì)胞增殖指數(shù)下降,細(xì)胞凋亡率減少,Wnt1/β-catenin信號通路抑制。結(jié)論:1.胃粘膜上皮癌變過程中發(fā)生了蛋白質(zhì)表達譜的改變,本研究鑒定出相關(guān)蛋白質(zhì)243個。2.聯(lián)合檢測PRSS1、HSP90α/β、TGM2、SerpinA3和P180蛋白質(zhì)表達,可為胃癌早期診斷提供參考。3.PRSS1通過影響Wnt1/β-catenin信號通路,調(diào)節(jié)細(xì)胞的生長與增殖、遷移及侵襲。
[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.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R735.2
本文編號：2232495
[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.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R735.2
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