Somatostatin receptor 1, a novel EBV-associated CpG hypermethylated gene, contributes to the pathogenesis of EBV-associated gastric cancer.

研究背景及目的：EB病毒(EBV)相關性胃癌的發病率約占胃癌的10%。近年來，越來越多的研究表明， EBV相關性胃癌的腫瘤抑制基因發生異常甲基化。然而，EBV的感染對全基因組DNA甲基化的影響尚不清楚。本研究通過分析EBV感染的細胞中全基因組DNA甲基化的情況，篩選出因EBV感染而發生甲基化的基因，並闡明靶基因在胃癌發生過程的作用。方法：本研究應用穩定轉染EBV的胃癌細胞AGS (AGS-EBV)和無EBV轉染的AGS細胞為模型。採用高解析度的甲基化DNA免疫共沉澱晶片技術(MeDIP-chip)比較AGS-EBV 和AGS全基因組DNA甲基化的變化，並根據基因本體論(GO)，對EBV誘導的甲基化基因進行分類。採用RT-PCR，去甲基化處理及亞硫酸氫鈉測序(BGS)等方法驗證EBV誘導的甲基化基因。同時，採用基因敲除和過表達方法體外研究靶基因的生物學功能：通過細胞活力實驗和集落形成實驗判斷靶基因對細胞增殖的影響；通过流式细胞技术、伤口愈合实验及侵袭实验研究筛选到的靶基因生长抑素受体1（SSTR1）的功能；此外，还通過腫瘤通路基因PCR晶片分析靶基因調控的下游腫瘤相關基因。结果：EBV編碼的小RNA(EBER)原位雜交方法和EBV潛伏期膜蛋白(LMP2A)的表達均證實AGS-EBV細胞中確實存在EBV的感染。和AGS細胞相比，發現AGS-EBV細胞中DNA甲基轉移酶3b(DNMT3b)的表達和活性顯著增加。AGS細胞中， LMP2A過表達後，DNMT3b的表達和活性也顯著增加。通過MeDIP-chip篩選出AGS-EBV中1,065甲基化有差異的基因，其中886基因為高甲基化。GO分析結果表明這些高甲基化基因參與KEGG信號通路。其中，六個新的高甲基化基因(MDGA2, IL15RA, SCARF2, EPHB6, SSTR1 和 REC8)在AGS-EBV細胞中的表達低於AGS；經過去甲基化處理之後，這些基因的表達水準有顯著增加。通過深入研究生長抑素受體1(SSTR1)的生物學功能，發現：敲除SSTR1 能促進胃癌細胞的增殖和集落形成；通過调节G1/S期的調節因子，加快細胞進入S期，顯著增加S期的細胞數目。此外，胰腺癌細胞PANC1細胞中，SSTR1的過表達，也進一步證實SSTR1確實是一種腫瘤抑制基因。腫瘤通路基因PCR晶片結果顯示SSTR1通過促進細胞週期抑制因數(包括p15，p16，p21和p27)的表達，同時抑制CDC25A 和Myc的表達，發揮抑制增殖作用，導致細胞週期停滯在G1期，減少細胞增殖。SSTR1也通過減少凋亡相關基因的表達參與細胞凋亡的過程。此外，SSTR1還能顯著下調遷移相關基因的表達。這些結果表明，在EB病毒相關胃癌的發生過程中，SSTR1通過調節細胞週期、凋亡及遷移的有關基因，進而抑制細胞增殖，減少細胞的遷移和轉移。结论：AGS感染EBV後，通過LMP2A促進DNMT3b的表達，激活DNMT3b的活性，導致886個腫瘤相關基因发生甲基化。SSTR1是一種EBV誘導的新的甲基化基因，在胃癌中具有抑制腫瘤的特性。研究表明， 由EBV誘導的SSTR1所具有的表觀遺傳學抑制作用參與EB病毒相關性胃癌的發病機制。Background and Aims: Epstein-Barr virus (EBV)-associated gastric cancer (GC) accounts for about 10% of all GCs. Accumulating evidences revealed aberrant rmethylation of tumor suppressor genes in EBV-associated GCs. However, the effect of EBV infection on the genome-wide aberrant DNA methylation remains unclear. We aim to profile the genome-wide EBV-associated hypermethylation in EBV-infected cells, to identify EBV-associated methylated genes and to elucidate their function in gastric carcinogenesis.Methods: The cell model of gastric cancer AGS cells with or without stable EBV infection was used in this study. Genome-wide DNA methylation profiles were compared between AGS-EBV and AGS cells by high resolution Methyl-DNA immunoprecipitation microarry (MeDIP-chip) assay. EBV-associated methylated genes were classified according to gene ontology (GO). The novel EBV-associated methylated candidates were validated using bisulfite genomic sequencing (BGS), RT-PCR, and demethylation treatment. Biological function of one of the candidate genes (Somatostatin Receptor 1, SSTR1) was studied in vitro using gene knockdown and over-expression approaches simultaneously. Effects of SSTR1 expression on gastric cancer cell was measured by cell viability assay, colony formation assay, flow cytometry, wound-healing assay and invasion assay. Gene modulation by SSTR1 in human cancer pathways was assessed by cancer pathway PCR array.Results: EBV infection was confirmed by EBER in situ hybridization. LMP2A expression was detected in AGS-EBV cells but not in EBV negative AGS cells. Expression and activity of DNMT3b was found to be significantly increased in AGS-EBV cells compared to AGS cells. Ectopic expression of LMP2A in AGS enhanced the expression and activity of DNMT3b. MeDIP-chip profiling identified a total of 1,065 genes differentially methylated by EBV infection (fold changes ≥2, P < 0.05) (fold-changes 2.4365.2). Gene ontology analysis indicated the enrichment of hypermethylated genes involving in important KEGG pathways. Notably, in addition to higher methylation levels confirmed by BGS, six novel hypermethylated genes (MDGA2, IL15RA, SCARF2, EPHB6, SSTR1 and REC8) were down-regulated in AGS-EBV cells as compared with AGS cells. Furthermore, demethylation treatment increased transcription levels of the six genes in AGS-EBV cells.The biological function of SSTR1 gene was further investigated. Knockdown of SSTR1 in GC cells increased cell proliferation (P < 0.05) and colony formation ability (P < 0.01), and markedly increased cells in S phase through regulating G1/S phase mediators. Overexpression of SSTR1 in PANC1 cell line further confirmed that SSTR1 indeed was a tumor suppressor gene. Analysis of SSTR1 regulation of cancer pathway demonstrated that SSTR1 exerted antiproliferative effect by inducing cyclin-dependent inhibitors (p15, p16, p21 and p27) and inhibiting cell divison cycle 25 homolog A (CDC25A) and Myc, resulting in cell cycle arrest in G1 phase and reduction of cell proliferation. SSTR1 also took part in proliferation by decreasing expression of apoptosis regulators. Moreover, SSTR1 significantly downregulated the expression of migration-related genes, including ITGA1, ITGA2, ITGA3, ITGB5, IL8, MMP1 and PLAUR. These findings suggest that SSTR1 inhibits proliferation and reduces cell migration/invasion in gastric cancer by deregulating genes invloved in the regulation of cell cycle, survival/apoptosis and migration.Conclusions: EBV infection in AGS cells induces genome-wide aberrant hypermethylation of 886 genes which involved in important cancer-related pathways. EBV-associated methylation is mediated by activation of DNMT3b through LMP2A. We identified and functionally characterized a novel EBV-associated methylated gene SSTR1 which exerted anti-tumor properties in GC. Epigenetic silencing of SSTR1 associated with EBV infection contributes to the pathogenesis of EBV-associated GC.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Zhao, Junhong.Thesis (Ph.D.)--Chinese University of Hong Kong, 2012.Includes bibliographical references (leaves 129-141).Abstract also in Chinese.ABSTRACT --- p.i摘 要 --- p.ivAcknowledgements --- p.viPublications --- p.viiResearch articles --- p.viiConference abstracts --- p.viiiTable of contents --- p.xlist of tables --- p.xiiilist of figures --- p.xivlist of abbreviations --- p.xviChapter Chapter 1 --- Introduction --- p.1Chapter 1.1 --- General introduction --- p.1Chapter 1.2 --- Gastric Cancer --- p.3Chapter 1.2.1 --- Eipdemiology of Gastric Cancer --- p.4Chapter 1.2.2 --- Pathology of Gastric Cancer --- p.8Chapter 1.2.3 --- Risk Factors for Gastric Cancers --- p.11Chapter 1.3 --- Epstein-Barr Virus-associated Gastric Cancer (EBVaGC) --- p.15Chapter 1.3.1 --- Historical Discovery and Harm of EBV --- p.15Chapter 1.3.2 --- Molecular Biology of EBV --- p.16Chapter 1.3.3 --- Latent and Lytic Infection of EBV --- p.18Chapter 1.3.4 --- EBV Products --- p.18Chapter 1.3.5 --- EBV-associated gastric cancer (EBVaGC) --- p.28Chapter 1.4 --- EBV-induced Epigenetic Alteration in Gastric Carcinogenesis --- p.36Chapter 1.4.1 --- Cytosine Methylation and CpG Island --- p.36Chapter 1.4.2 --- DNA Methylation in Gastric Cancer --- p.39Chapter 1.5 --- How to identify EBV-induced promoter methylation in gastric cancer --- p.45Chapter 1.5.1 --- Methylated DNA Immunoprecipitation (MeDIP) --- p.48Chapter 1.5.2 --- Combined Bisulfite Restriction Analysis (COBRA) --- p.49Chapter 1.5.3 --- Bisulfite Genomic Sequencing --- p.49Chapter 1.5.4 --- Pyrosequencing --- p.49Chapter Chapter 2 --- Materials and Methods --- p.51Chapter 2.1 --- Materials --- p.51Chapter 2.1.1 --- Cancer Cell Lines and Culture Condition --- p.51Chapter 2.1.2 --- Primary GC Samples --- p.52Chapter 2.2 --- EBV Encoded Nuclear RNA (EBER) in situ Hybridization (EBER-ISH) --- p.52Chapter 2.3 --- Western Blot Analysis --- p.53Chapter 2.4 --- Plasmid and Transfection --- p.56Chapter 2.4.1 --- Plasmid Construction and Extraction --- p.56Chapter 2.4.2 --- Plasmid Transfection --- p.59Chapter 2.5 --- Gene Expression Analysis --- p.59Chapter 2.5.1 --- Purification of Total RNA (RNeasy Kit, Qiagen) --- p.59Chapter 2.5.2 --- cDNA Reverse Transcription --- p.60Chapter 2.5.3 --- Semi-Quantitative PCR --- p.62Chapter 2.5.4 --- Quantitative Real-Time PCR (qRT-PCR) --- p.64Chapter 2.6 --- DNMT1 and 3b Activity Assay --- p.64Chapter 2.7 --- DNA Methylation Analysis --- p.64Chapter 2.7.1 --- Genomic DNA Extraction --- p.64Chapter 2.7.2 --- Genome-wide Profiling of EBV-associated DNA Methylation by MeDIP-chip --- p.65Chapter 2.7.3 --- Bioinformatics Analysis --- p.66Chapter 2.7.4 --- CpG Island Prediction and Analysis of the Targets’ Promoter Region --- p.66Chapter 2.7.5 --- DNA Sodium Bisulfite Modification --- p.67Chapter 2.7.6 --- Target Gene Methylation in GC Cell Lines --- p.67Chapter 2.7.7 --- Bisulfite Pyrosequencing Analysis in GC Tissue Samples --- p.70Chapter 2.8 --- Biological Function Analysis of SSTR1 --- p.72Chapter 2.8.1 --- Cell Proliferation Assay for Stable Transfection --- p.72Chapter 2.8.2 --- Colony Formation Assay --- p.72Chapter 2.8.3 --- Cell Cycle Analysis Assay --- p.72Chapter 2.8.4 --- Cell Migration Analysis --- p.73Chapter 2.8.5 --- Invasion Analysis --- p.73Chapter 2.8.6 --- Human Cancer Pathway Finder RT2 Profiler PCR Array Analysis --- p.74Chapter 2.9 --- Statistical Analysis --- p.76Chapter Charpter 3 --- results --- p.77Chapter 3.1 --- EBV Infection in AGS-EBV Cell Model --- p.77Chapter 3.2 --- Activation of DNMT3b in AGS-EBV Cells --- p.80Chapter 3.3 --- LMP2A Induced DNMT3b Activity in AGS Cells --- p.82Chapter 3.4 --- Genome-wide Profiling of DNA Methylation Associated with EBV Infection Using MeDIP-chip --- p.84Chapter 3.5 --- EBV-associated Cancer Pathways Defined by EBV-associated Promoter Methylated Genes --- p.86Chapter 3.6 --- CpG Hypermethylation and Transcriptional Silencing of EBV-associated Methylated Genes in AGS-EBV Cells --- p.88Chapter 3.7 --- Bioinformatics Analysis of SSTR1 Using University of California Santa Cruz Genome Bioinformatics (UCSC) Database and CpG Island Searcher --- p.93Chapter 3.8 --- COBRA Analysis of SSTR1 Promoter Methylation in GC Cell Lines --- p.93Chapter 3.9 --- Frequent SSTR1 Hypermethylation was Associated with EBV Positive Primary Gastric Cancer --- p.96Chapter 3.10 --- SSTR1 was Down-regulated in GC Cell Lines through RNA Interference --- p.103Chapter 3.11 --- SSTR1 Knockdown Induced Cell Proliferation in GC Cell Lines --- p.105Chapter 3.12 --- SSTR1 Knock-down Promoted Cells to Enter into S Phase --- p.108Chapter 3.13 --- SSTR1 Knock-down Increased the Migration Ability of GC --- p.110Chapter 3.14 --- SSTR1 Knock-down Promoted Cell Invasion --- p.112Chapter 3.15 --- Ectopic Expression of SSTR1 Inhibited Proliferation and Clonogenicity in PANC1 Cancer Cells --- p.114Chapter 3.16 --- Identification of Genes Modulated by SSTR1 --- p.116Chapter Chapter 4 --- Discussion --- p.119Chapter Chapter 5 --- Limitation of the study --- p.127ConclusionS --- p.128Reference --- p.12