Cervical cancer is the third leading cause of cancer death in women worldwide. Although cervical cancer is commonly infected with human papillomavirus (HPV), HPV infection alone is insufficient to induce malignant changes. Many characteristic genetic and epigenetic alterations have been identified in invasive cervical carcinomas but relatively little is known about the specific genetic and molecular alterations that allow pre-invasive epithelial cells to acquire the ability to progress to invasive squamous cell carcinomas. Recently, a family of small non-coding RNAs termed microRNAs (miRNAs) with specific inhibitory functions on target gene expression has been suggested to play an important role in the pathogenesis of human cancers including lung and breast cancer but remain undefined in cervical cancer. / Genome wide chromosomal copy number changes in cervical cancer by Agilent high-density array Comparative Genomic Hybridization demonstrated that only a very limited number of genomic imbalances have an impact on the miRNA profile in cervical cancer cells, although a high proportion of genomic loci containing miRNA genes exhibited DNA copy number alterations in other cancers. The impact of the genomic aberration on their mRNA expression was then confirmed by Aligent Whole Human Genome gene expression array. This suggests that the regulation of miRNA and mRNA expression may be different in cervical cancer. / In conclusion, our global miRNA profiling identified the common differentially expressed and genomic aberration independent miRNAs in cervical cancer. We further revealed the inhibition of hsa-miR-182 reduced tumor cell growth in vitro and in vivo through apoptosis and cell cycle mechanism. This provides new evidence that hsa-miR-182 may contribute to the pathogenesis of cervical cancer. / Keywords. MicroRNA, Cervical Cancer, Tumor Growth / To identify microRNA(s) associated with the tumorigenesis of cervical cancer, we firstly used the TaqMan MicroRNA Assays to survey and quantify a panel of 157 known human miRNAs in cervical cancer cell fines and micro-dissected normal cervical epithelium cells. We identified 2 microRNAs that were differentially up-regulated (fold change > 2, p < 0.05) and 9 differentially down-regulated (fold change > 2, p < 0.05) in cervical cancer cell lines comparing with normal cervical epithelium. Further investigation in tumor samples confirmed these two up-regulated miRNAs (hsa-miR-182 and -183 ) and 3 down-regulated miRNA (hsa-miR-145, 150, 195) from 4 investigated downregulated miRNAs (hsa-miR-145, 150, 195 and 328). / To investigate the biological function of those aberrantly expressed microRNAs, we chose one of the most aberrantly up-regulated microRNA ( hsa-miR-182, fold change > 10) for further investigation. Inhibition of hsa-miR-182 by antisense oligonucleotides inhibited HeLa cervical cancer cell growth in vitro and reduced tumor cell volume in vivo. Gene expression array analysis of HeLa cells with hsa-miR-182 knockdown and over-expression showed specific hsa-miR-182 targeting pathway in apoptosis and cell cycle. It indicated the roles of hsa-miR-182 in cervical cancer growth through apoptosis and cell cycle functions. / Tang, Tao. / Adviser: Richard K W Choy. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3446. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 155-169). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Gastric cancer is the second leading cause of cancer death worldwide and in China. The mechanism of gastric carcinogenesis is not fully understood. Epigenetic studies indicated that inactivation of tumor suppressor genes by DNA hypermethylation plays a crucial role in the progression of gastric cancer. Epigenetic inactivation of secreted frizzled-related protein (SFRP 1) by methylation plays a pivotal role on the development of various cancers. However, the role of SFRP family genes in gastric cancer remains largely unknown. We aimed to characterize the epigenetic abnormalities and discover novel biomarkers for early detection of gastric cancer. We investigated the epigenetic alterations in gastric adenocarcinoma by microarray based analysis and gene promoter hypermethylation. Based of the microarray data, we determined the functional significance and frequency of SFRP family genes hypermethylation in human gastric cancer. We screened the mRNA expression and methylation status of the SFRP family members in human gastric cancer cell lines and primary gastric cancer samples. Demethylation study of SFRP family genes were done by treating gastric cancer cell lines with 5'Aza. The biological effects of SFRP were analyzed by flow cytometry, cell viability assay and tumor growth in nude mice. SFRP1, 2, 4 and 5 were undetectable in 100% (7/7), 100% (7/7), 42.8% (3/7) and 85.7% (6/7) of gastric cancer cell lines, respectively. However, only SFRP2 showed significant down-regulation in gastric cancer compared with adjacent non-cancer samples (P<0.01). Treatment with demethylation agent, 5'-Aza, restored the expression of SFRP2 in all 7 cancer cell lines. Promoter hypermethylation of SFRP2 was detected in 73.3% of primary gastric cancer samples and 20% of adjacent non-cancer tissue (P<0.01). Bisulfite sequencing confirmed the density of promoter methylation in cell line, primary gastric cancer tissue and their adjacent non-cancer tissue. Transfection of SFRP2 induced cell apoptosis, inhibited proliferation in vitro and suppressed tumor growth in vivo. Furthermore, SFRP2 methylation was detected in 37.5% of samples showing intestinal metaplasia. Methylated SFRP2 was also detected in 66.7% of serum samples from cancer patients but not in normal controls. Epigenetic inactivation of SFRP2, but not SFRP1, SFRP4 and SFRP5 is a common and early event of carcinogenesis. Hence, detection of SFRP2 methylation in serum may have diagnostic value in gastric cancer patients. / by Cheng, Yuen Yee. / Adviser: FKL Chan. / Source: Dissertation Abstracts International, Volume: 69-02, Section: B, page: 0803. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 165-179). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / School code: 1307.

A case-control study was previously carried out to identify HCC-associated genomic markers on HBV. Some of them are clustered at the preS1 and X promoter regions of HBV genotype B and core promoter of HBV subgenotype Cs. The functional significance of these markers to the virus was investigated in our study. Our result showed that one of those markers, the G1613A mutation on core promoter, can significantly increase the promoter activity in a genotype-dependent manner and the effect is reversible by the A-to-G back mutation. We have established an in vitro full-length HBV genome transfection system and the result suggested that the G1613A mutation suppressed the e antigen (HBeAg) secretion and enhanced virus DNA production by downregulating the precore (preC) mRNA transcription. In consistence to the clinical study, the mutation was associated to serum HBV DNA level higher than 6 log copies/1M in female HBV carriers in a univariate analysis. In addition, we demonstrated that the G1613A mutation is a hot spot mutation situated on the negative regulatory element (NRE) on the core promoter in an alignment analysis. To further investigate the molecular mechanism of the mutation, two unknown protein complexes had been shown to bind on the NRE. They showed different binding affinity to the G1613-wild-type and A1613-mutant NRE sequence. Moreover, we showed that in vitro synthesized RFX1 protein could bind to the mutated NRE probe at a higher affinity than that to wild-type NRE probe. Overall, our result suggests that the G1613A mutation exerts its effect by differential binding to some proteins via the NRE region. Studying the mechanism of the mutations may provide insights to the viral pathogenesis and HBV-associated HCC, which has long been a health burden in Asia-Pacific countries. / Infection of hepatitis B virus (HBV) causes acute and chronic hepatitis and is closely associated with the development of cirrhosis and hepatocellular carcinoma (HCC). Approximately 60-80% of world's HCC is related to HBV, and it is the third most common cause of cancer death in Asia-Pacific region. Almost 400 million people are chronically infected with HBV and one-third was likely to die of complications of cirrhosis, including liver failure and HCC. As there is a shortage of effective curative treatments, detection and prognosis of the risk of cancer development will be essential to improve survival of patients with chronic HBV infection. / Li, Man Shan. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 198-210). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Background. B-lymphoma Moloney murine leukemia virus insertion region-1 (Bmi-1) acts as an oncogene in various cancer such as non-small cell lung cancer, colon cancer, gastric cancer, bladder cancer and nasopharyngeal cancer (NPC). / Methods. Immunohistochemistry was performed to evaluate Bmi-1 expression in 252 breast cancer samples. The correlations were analyzed between Bmi-1 expression and clinicopathologic parameters, including age, tumor size, lymph nodal involvement, distant metastasis, clinical stages, hormone receptor (ER, PR) and Human Epidermal Growth Factor Receptor 2 (HER-2). The overall survivals were compared by Kaplan-Meier analysis based on Bmi-1 expression. / Results. Bmi-1 expression was significantly increased in primary cancer tissues than in matched adjacent non-cancerous tissues ( P<0.001). Only 35.9% (14 of 39) of adjacent non-cancerous tissues displayed high expression compared with 72.2% (182 of 252) in primary cancer tissues. Among adjacent non-cancerous tissues, no Bmi-1 staining signal was detected in 30.8% (12 in 39) samples. Only 28.2% (11 in 39) samples showed nucleus staining and the remaining 41.0% (16 in 39) samples exhibited cytoplasm staining. Of those cancer tissues, however, 75.4% (190 in 252) was stained in the nucleus and 24.6% (62 in 252) located in the cytoplasm. The elevated Bmi-1 expression was correlated with advanced clinicopathologic classifications (T, N, M) and clinical stages (P<0.001, respectively). A high level of Bmi-1 expression displayed unfavorable overall survival ( P<0.001). The overall survival rate, assessed by the Kaplan-Meier method, was 85.1% (57 in 67) in low Bmi-1 expression group, whereas it was only 59.9% (103 in 172) in high Bmi-1 expression group. In addition, Bmi-1 serves as a high risk for breast cancer and the relative risk increased almost four fold in patients with high Bmi-1 expression compared with that with low Bmi-1 expression by univariate Cox regression analyses. After the adjustment of the confounding factors, Bmi-1 was still found to predict the poor survival (P=0.042), which indicated Bmi-1 was an independent prognostic factor. The overexpression of Bmi-1 increased the mobility and invasiveness in 76N-TERT and MCF-10A, concurrent EMT-like molecular changes, the stabilization of Snail protein and the activation of Akt/GSK3beta pathway. Consistent with these observations, the repression of Bmi-1 in MDA-MB-435S remarkably attenuated the cellular mobility, invasiveness and transformation, as well as tumorigenesis and spontaneous lung metastases in nude mice. In addition, the repression of Bmi-1 reversed the EMT markers and inhibited the Akt/GSK3beta/Snail pathway. However, ectopic Bmi-1 alone was not able to lead to the phenotype of HMECs. Additionally, discordant mRNA expression levels of Bmi-1 and E-cadherin were detected between primary cancer tissues and matched adjacent non-cancerous tissues. The mRNA level of Bmi-1 was strongly up-regulated in breast cancer tissues compared with paired non-cancerous tissues ( P=0.001), whereas the mRNA level of E-cadherin was markedly down-regulated (P=0.042). Furthermore, there was a converse correlation between Bmi-1 and E-cadherin expression at the transcriptional level ( P=0.041). (Abstract shortened by UMI.) / Guo, Baohong. / Adviser: Kung, Hsiang Fu. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 161-183). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

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.iv / Acknowledgements --- p.vi / Publications --- p.vii / Research articles --- p.vii / Conference abstracts --- p.viii / Table of contents --- p.x / list of tables --- p.xiii / list of figures --- p.xiv / list of abbreviations --- p.xvi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- General introduction --- p.1 / Chapter 1.2 --- Gastric Cancer --- p.3 / Chapter 1.2.1 --- Eipdemiology of Gastric Cancer --- p.4 / Chapter 1.2.2 --- Pathology of Gastric Cancer --- p.8 / Chapter 1.2.3 --- Risk Factors for Gastric Cancers --- p.11 / Chapter 1.3 --- Epstein-Barr Virus-associated Gastric Cancer (EBVaGC) --- p.15 / Chapter 1.3.1 --- Historical Discovery and Harm of EBV --- p.15 / Chapter 1.3.2 --- Molecular Biology of EBV --- p.16 / Chapter 1.3.3 --- Latent and Lytic Infection of EBV --- p.18 / Chapter 1.3.4 --- EBV Products --- p.18 / Chapter 1.3.5 --- EBV-associated gastric cancer (EBVaGC) --- p.28 / Chapter 1.4 --- EBV-induced Epigenetic Alteration in Gastric Carcinogenesis --- p.36 / Chapter 1.4.1 --- Cytosine Methylation and CpG Island --- p.36 / Chapter 1.4.2 --- DNA Methylation in Gastric Cancer --- p.39 / Chapter 1.5 --- How to identify EBV-induced promoter methylation in gastric cancer --- p.45 / Chapter 1.5.1 --- Methylated DNA Immunoprecipitation (MeDIP) --- p.48 / Chapter 1.5.2 --- Combined Bisulfite Restriction Analysis (COBRA) --- p.49 / Chapter 1.5.3 --- Bisulfite Genomic Sequencing --- p.49 / Chapter 1.5.4 --- Pyrosequencing --- p.49 / Chapter Chapter 2 --- Materials and Methods --- p.51 / Chapter 2.1 --- Materials --- p.51 / Chapter 2.1.1 --- Cancer Cell Lines and Culture Condition --- p.51 / Chapter 2.1.2 --- Primary GC Samples --- p.52 / Chapter 2.2 --- EBV Encoded Nuclear RNA (EBER) in situ Hybridization (EBER-ISH) --- p.52 / Chapter 2.3 --- Western Blot Analysis --- p.53 / Chapter 2.4 --- Plasmid and Transfection --- p.56 / Chapter 2.4.1 --- Plasmid Construction and Extraction --- p.56 / Chapter 2.4.2 --- Plasmid Transfection --- p.59 / Chapter 2.5 --- Gene Expression Analysis --- p.59 / Chapter 2.5.1 --- Purification of Total RNA (RNeasy Kit, Qiagen) --- p.59 / Chapter 2.5.2 --- cDNA Reverse Transcription --- p.60 / Chapter 2.5.3 --- Semi-Quantitative PCR --- p.62 / Chapter 2.5.4 --- Quantitative Real-Time PCR (qRT-PCR) --- p.64 / Chapter 2.6 --- DNMT1 and 3b Activity Assay --- p.64 / Chapter 2.7 --- DNA Methylation Analysis --- p.64 / Chapter 2.7.1 --- Genomic DNA Extraction --- p.64 / Chapter 2.7.2 --- Genome-wide Profiling of EBV-associated DNA Methylation by MeDIP-chip --- p.65 / Chapter 2.7.3 --- Bioinformatics Analysis --- p.66 / Chapter 2.7.4 --- CpG Island Prediction and Analysis of the Targets’ Promoter Region --- p.66 / Chapter 2.7.5 --- DNA Sodium Bisulfite Modification --- p.67 / Chapter 2.7.6 --- Target Gene Methylation in GC Cell Lines --- p.67 / Chapter 2.7.7 --- Bisulfite Pyrosequencing Analysis in GC Tissue Samples --- p.70 / Chapter 2.8 --- Biological Function Analysis of SSTR1 --- p.72 / Chapter 2.8.1 --- Cell Proliferation Assay for Stable Transfection --- p.72 / Chapter 2.8.2 --- Colony Formation Assay --- p.72 / Chapter 2.8.3 --- Cell Cycle Analysis Assay --- p.72 / Chapter 2.8.4 --- Cell Migration Analysis --- p.73 / Chapter 2.8.5 --- Invasion Analysis --- p.73 / Chapter 2.8.6 --- Human Cancer Pathway Finder RT2 Profiler PCR Array Analysis --- p.74 / Chapter 2.9 --- Statistical Analysis --- p.76 / Chapter Charpter 3 --- results --- p.77 / Chapter 3.1 --- EBV Infection in AGS-EBV Cell Model --- p.77 / Chapter 3.2 --- Activation of DNMT3b in AGS-EBV Cells --- p.80 / Chapter 3.3 --- LMP2A Induced DNMT3b Activity in AGS Cells --- p.82 / Chapter 3.4 --- Genome-wide Profiling of DNA Methylation Associated with EBV Infection Using MeDIP-chip --- p.84 / Chapter 3.5 --- EBV-associated Cancer Pathways Defined by EBV-associated Promoter Methylated Genes --- p.86 / Chapter 3.6 --- CpG Hypermethylation and Transcriptional Silencing of EBV-associated Methylated Genes in AGS-EBV Cells --- p.88 / Chapter 3.7 --- Bioinformatics Analysis of SSTR1 Using University of California Santa Cruz Genome Bioinformatics (UCSC) Database and CpG Island Searcher --- p.93 / Chapter 3.8 --- COBRA Analysis of SSTR1 Promoter Methylation in GC Cell Lines --- p.93 / Chapter 3.9 --- Frequent SSTR1 Hypermethylation was Associated with EBV Positive Primary Gastric Cancer --- p.96 / Chapter 3.10 --- SSTR1 was Down-regulated in GC Cell Lines through RNA Interference --- p.103 / Chapter 3.11 --- SSTR1 Knockdown Induced Cell Proliferation in GC Cell Lines --- p.105 / Chapter 3.12 --- SSTR1 Knock-down Promoted Cells to Enter into S Phase --- p.108 / Chapter 3.13 --- SSTR1 Knock-down Increased the Migration Ability of GC --- p.110 / Chapter 3.14 --- SSTR1 Knock-down Promoted Cell Invasion --- p.112 / Chapter 3.15 --- Ectopic Expression of SSTR1 Inhibited Proliferation and Clonogenicity in PANC1 Cancer Cells --- p.114 / Chapter 3.16 --- Identification of Genes Modulated by SSTR1 --- p.116 / Chapter Chapter 4 --- Discussion --- p.119 / Chapter Chapter 5 --- Limitation of the study --- p.127 / ConclusionS --- p.128 / Reference --- p.129

研究背景和目的 / 肝癌是非常高死亡率的恶性肿瘤之一。由于传统化疗方式的局限性，表观遗传治疗方法可能成为肝癌治疗的替代方法。研究报道ZBP-89诱导肝癌细胞Bak的表达，表观调控是否参与该诱导作用，目前仍然不清楚。 / HDAC3被认为是化疗靶点和肝癌复发的肿瘤标记物。它常常在肝癌组织中高表达，对HDAC3的抑制作用可以增加肝癌的化疗效果。我们的研究表明ZBP-89可以降低肝癌细胞HDAC3的表达，但机制未明。蛋白的翻译后调控是细胞生化过程的重要调节因素。所以，研究调节HDAC3的降低途径对肝癌的发生和复发具有非常重要的研究意义。 / 本研究旨在研究ZBP-89调控Bak表达的表观遗传机制。同时，弄清楚DNA甲基化转移酶和组蛋白去乙酰化酶是否参与ZBP-89对Bak的调控作用，进一步阐明ZBP-89对HDAC3降低通路的机制。 / 方法和结果 / 肝癌病人组织蛋白分析表明，相对于癌旁组织，肝癌组织Bak和ZBP-89蛋白表达降低，而DNMT1和HDAC3表达升高。免疫共沉淀技术显示ZBP-89与HDAC3、 DNMT1结合，但不与HDAC4， DNMT3a和DNMT3b结合。相应地，HDAC3和 DNMT1免疫沉淀分析也显示三者形成免疫复合物。我们在肝癌细胞中过表达ZBP-89，验证它会不会影响HDACs和DNMTs的活性。实验结果表明过表达的ZBP-89抑制HDACs和DNMTs的活性。进一步发现ZBP-89调节的Bak表达可能是通过抑制HDACs活性和维持组蛋白H3和H4乙酰化水平实现的。另一方面，我们同样证明HDAC的抑制剂（HDACi）VPA和TSA可以诱导肝癌细胞Bak表达，此外，siRNA干扰HDAC3的表达同样可以诱导Bak表达。 / 对DNMT1表达的抑制和使用DNMT抑制剂（DNMTi）Zebularine也可以诱导Bak的表达。染色质免疫沉淀结果显示ZBP-89结合于Bak的启动子区域，从-3188bp到-3183bp，从-275到-49。 ZBP-89可以抑制DNMT的活性，那么ZBP-89是否会影响DNA中CpG岛甲基化状态和甲基化结合蛋白（MeCP2）的结合能力，这一点仍需要进一步证实。结果表明ZBP-89可以抑制MeCP2结合基因组DNA。为进一步揭示MeCP2是否由于启动子区域CpG岛去甲基化影响其结合能力，我们采用亚硫酸盐测序方法。测序结果显示ZBP-89过表达可以影响Bak启动子CpG岛的甲基化状态，并促进其去甲基化。 / 腺病毒介导的ZBP-89过表达降低HDAC3表达呈现剂量依赖性，然而HDAC3 的mRNA水平并没有受到ZBP-89的表达。免疫共沉淀方法和蛋白免疫印迹实验用于分析Pin1和HDAC3复合物，磷酸化IκB和HDAC3复合物的结合情况。结果表明Pin1结合HDAC3并促进HDAC3的减少。同时，HDAC3与磷酸的IκB结合并进入蛋白减少途径。 / 构建的mU6-siPin1表达质粒用于敲除肝癌细胞Pin1的表达，方法检测基因表达水平。Pin1的缺失表达阻碍ZBP-89介导的HDAC3降低。在Pin1 敲除细胞系 JB6 C141 Pin1⁻/⁻ 和Pin1过表达细胞系的研究，ZBP-89更加能促进Pin1⁺/⁺细胞中HDAC3减少，而对Pin1⁺/⁺的细胞则没那么明显。由此肯定了Pin1在ZBP-89介导的HDAC3降低中的重要作用。进一步研究发现， IκB激酶 (IKK)抑制剂，CAY10576，能抑制 ZBP-89介导的HDAC3的降低；而SN50, p65/p50人核抑制多肽，则不影响HDAC3的降低。研究结果证明HDAC3的降低依赖IκB通路，而不是NF- κB活性。 / 我们用人肝癌细胞的裸鼠移植瘤模型研究ZBP-89调控Bak表达的表观遗传机制，及其对肝癌的治疗效果。研究结果表明ZBP-89蛋白和组蛋白抑制剂VPA和DNA甲基化抑制zebularine都能抑制肿瘤的生长，并诱导肿瘤组织Bak表达及细胞凋亡。VPA和zebularine联合治疗的效果更好。研究也表明ZBP-89可以在体内降低HDAC3蛋白水平。 / 结论 / 本研究揭示了ZBP-89调节Bak蛋白表达和肝癌细胞凋亡的表观遗传机制。同时，进一步揭示ZBP-89联合Pin1经由IκB通路调节HDAC3降低的机制. 本研究为肝癌表观遗传学的治疗提供研究基础和科学依据。 / Background / Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies worldwide with a very high mortality. Because the success of the conventional therapies is limited, epigenetic therapy may represent an alternative for HCC management. ZBP-89 is known to induce Bak in HCC. However, it is unclear whether epigenetic mechanisms contribute to ZBP-89-mediated Bak. / Histone acetylase 3 (HDAC3) is realized as a chemotherapy target and a biomarker of recurrence in HCC. HDAC3 is frequently overexpressed in HCC and its inhibition enhances the efficacy of anti-HCC chemotherapy. The pilot data have indicated that ZBP-89 reduced HDAC3 in HCC but the mechanism responsible was unknown. The post-translational modification of proteins functions as a key regulatory factor in cellular physiological procedures, such as ubiquitinoylation degradation. As a biomarker of HCC development and recurrence, it is important to understand how ZBP-89 mediates the reduction of HDAC3. / This study focuses on if ZBP-89 regulates Bak expression through epigenetic mechanisms. It is designed to investigate whether DNA methyltransferases (DNMTs), histone acetylases (HDACs) are involved in regulation of ZBP-89-induced Bak expression. The study also elucidates the mechanism how ZBP-89 reduces the level of HDAC3 protein. / Methods and Results / The levels of Bak and ZBP-89 as shown on western blots were reduced but DNMT1 and HDAC3 were increased in HCC cancer tissues compared to the corresponding non-cancer tissues. Co-immunoprecipitation experiments showed that ZBP-89 bound to HDAC3 and DNMT1 but not other epigenetic enzymes, such as HDAC4, DNMT3a and DNMT3b. To clarify if ZBP-89 affects the activities of HDACs and DNMTs, ZBP-89 was overexpressed in HCC cells. Enzyme activities of HDACs and DNMTs were determined using relevant assay kits. Results showed that overexpressed ZBP-89 inhibited the activities of HDACs and DNMTs. Further experiments indicated that ZBP-89-mediated Bak up-regulation might contribute to maintenance of histone H3 and H4 acetylation through inhibition of HDACs activity. In another set of experiments, we also found an increased Bak expression in HCC cells when the cells were treated with HDAC inhibitors (HDACi) VPA and TSA. HDAC3 siRNA also increased Bak expression. / Both knockdown of DNMT1 expression and administration of DNMTs inhibitors (zebularine) induced Bak expression. Chromatin immunoprecipitation (ChIP) showed that ZBP-89 bound to Bak promoter at the region from -3188bp to -3183bp and from -275 to -49. As ZBP-89 inhibits DNMT activity, it is essential to know whether its inhibition affectes DNA CpG methylation status and methyl-CpG binding protein (MeCP) binding. The results showed that ZBP-89 overexpression inhibited MeCP2 binding to genomic DNA. The finding indicated that decreased MeCP2 binding to DNA might be due to decreased methyl-CpG number in Bak promoter, suggesting that ZBP-89 might affect CpG island methylation status. Therefore, the bisulfite modified DNA sequencing method was used to clarify if Bak promoter CpG island methylation status was altered after ZBP-89 overexpression. Results revealed that ZBP-89 overexpression could demethylate the CpG islands in Bak promoter. / ZBP-89 overexpression dose-dependently reduced the expression of HDAC3 at protein level but not at mRNA level. Co-immunoprecipitation and western blot methods were used to analyze Peptidyl-prolyl cis/trans isomerase 1 (Pin1) and HDAC3, phospho-I kappa B (pIκB), and the result revealed that HDAC3 could bound with either Pin1 or pIκB to promote the reduced expression of HDAC3. / Constructed mU6-siPin1 vector was used to knockdown Pin1 expression in HCC cells. We found that knockdown of Pin1 expression blocked ZBP-89-mediated HDAC3 reduction. Experiments performed in Pin1 allele-knockdown JB6 C141 Pin1⁻/⁻ and Pin1⁺/⁺ cells showed that the reduction of HDAC3 by ZBP-89 was greater in Pin1⁺/⁺ cells than in Pin1⁻/⁻ cells, confirming the role of Pin1 in ZBP-89-mediated HDAC3 reduction. Furthermore, the ZBP-89-mediated HDAC3 reduction was suppressed by CAY10576, an IκB kinase (IKK) activation inhibitor but not by SN50, a p65/p50 translocation inhibitor, suggesting that HDAC reduction may depend on IκB kinase rather than NF-κB activity. / HCC xenograft mouse model was used to support the involvement of epigenetic mechanism in ZBP-89-induced Bak expression and its therapeutic effects against HCC. Results showed that ZBP-89 as well as HDAC inhibitor valproic acid (VPA) or/and DNMT inhibitor zebularine stimulated Bak expression and induced apoptosis of tumor cells in an HCC xenograft mouse model, arresting tumor growth. In HCC xenografe model, treatment by injection of Ad-ZBP-89 viral expression vector mediated ZBP-89 expression decreased HDAC3 expression, but not HDAC4. / Conclusions / In conclusion, the study demonstrates a novel mechanism through which ZBP-89 mediates an epigenetic pathway to promote Bak expression, and induce apoptosis in HCC cells. It also reveals the mechanism of HDAC3 reduction by ZBP-89 is dependent on IκB, which requires the presence of Pin1. This pathway may help develop future epigenetic therapy against HCC. / 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. / 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. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Ye, Caiguo. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 123-140). / Abstracts also in Chinese. / Abstract --- p.i / 摘要 --- p.v / Publications --- p.viii / Acknowledgements --- p.ix / Abbreviations --- p.xi / List of Tables --- p.xiii / List of figures --- p.xiv / Chapter Chapter One: --- General Introduction --- p.1 / Chapter 1.1 --- Background --- p.2 / Chapter 1.2 --- The complexity of HDAC family and functions --- p.3 / Chapter 1.2.1 --- HDAC family --- p.4 / Chapter 1.2.2 --- Multifunction of HDACs --- p.6 / Chapter 1.3 --- HDACs and apoptosis --- p.6 / Chapter 1.3.1 --- HDAC regulates apoptotic-related gene expression --- p.9 / Chapter 1.3.2 --- HDACs regulate apoptosis through protein complexes --- p.18 / Chapter 1.3.3 --- HDACs mediates non-histone deacetylation and apoptosis --- p.21 / Chapter 1.3.4 --- HDACs degradation deficiency and apoptosis --- p.24 / Chapter 1.4 --- DNMTs and epigenetic modification --- p.25 / Chapter 1.4.1 --- DNMT family --- p.25 / Chapter 1.4.2 --- CpG islands methylation and HCC --- p.26 / Chapter 1.5 --- Perspectives --- p.28 / Chapter Chapter Two: --- ZBP-89 up-regulates Bak expression through inhibition the activity of HDACs and DNMTs --- p.30 / Chapter 2.1 --- Introduction --- p.31 / Chapter 2.2 --- Materials and Methods --- p.33 / Chapter 2.2.1 --- Hepatocellular carcinoma patient samples and cell lines --- p.33 / Chapter 2.2.2 --- Chemicals and reagents --- p.34 / Chapter 2.2.3 --- Cell proliferation --- p.34 / Chapter 2.2.4 --- Adenovirus infection of cells --- p.35 / Chapter 2.2.5 --- Apoptosis detection --- p.36 / Chapter 2.2.6 --- Transfection of siRNA and plasmid --- p.36 / Chapter 2.2.7 --- Co-immunoprecipitation (co-IP) --- p.37 / Chapter 2.2.8 --- Western blotting --- p.37 / Chapter 2.2.9 --- Immunohistochemistry and Immunofluorescence --- p.38 / Chapter 2.2.10 --- Chromatin immunoprecipitation --- p.38 / Chapter 2.2.11 --- Sodium bisulfite modified sequencing of Bak promoter --- p.40 / Chapter 2.2.12 --- Histone deacetylase activity assay --- p.41 / Chapter 2.2.13 --- DNA methyltransferases enzyme activity --- p.42 / Chapter 2.2.14 --- Xenograft animal model --- p.43 / Chapter 2.2.15 --- Statistical analysis --- p.43 / Chapter 2.3 --- Results --- p.45 / Chapter 2.3.1 --- ZBP-89 interacts with DNMT1 and HDAC3 --- p.45 / Chapter 2.3.2 --- DNA methyltransferase-1 and histone deacetylase 3 are overexpressed in cancer tissues --- p.48 / Chapter 2.3.3 --- Inhibition of HDACs and DNMTs induces Bak expression and apoptosis --- p.58 / Chapter 2.3.4 --- Adenovirus mediated ZBP-89 expression inhibits HDACs activity --- p.65 / Chapter 2.3.5 --- ZBP-89 suppresses DNMTs activity --- p.67 / Chapter 2.3.6 --- Overexpressed ZBP-89 demethylates methyl-CpG islands --- p.69 / Chapter 2.3.7 --- Downregulation of HDAC3 and DNMT1 enhances Bak expression --- p.74 / Chapter 2.3.8 --- Xenograft nude mouse model reveals that Ad-ZBP-89 adenovirus diminishes tumor volume and induces Bak expression and apoptosis --- p.75 / Chapter 2.4 --- Discussion --- p.81 / Chapter Chapter Three: --- ZBP-89 targets IkappaB to reduce HDAC3 via a Pin1-dependent pathway --- p.86 / Chapter 3.1 --- Introduction --- p.87 / Chapter 3.2 --- Materials and Methods --- p.89 / Chapter 3.2.1 --- Cell lines, chemicals and reagents --- p.89 / Chapter 3.2.2 --- Transfection of siRNA plasmid --- p.89 / Chapter 3.2.3 --- Plasmid extraction by mini-prep --- p.90 / Chapter 3.2.4 --- Co-immunoprecipitation (co-IP) and Western blotting --- p.91 / Chapter 3.2.5 --- Total RNA extraction --- p.92 / Chapter 3.2.6 --- Reverse transcription and real-time PCR --- p.93 / Chapter 3.2.7 --- Immunohistochemistry and Immunofluorescence --- p.94 / Chapter 3.2.8 --- Xenograft animal model --- p.95 / Chapter 3.2.9 --- Statistical analysis --- p.95 / Chapter 3.3 --- Results --- p.97 / Chapter 3.3.1 --- ZBP-89 overexpression diminishes HDAC3 expression but not HDAC4 --- p.97 / Chapter 3.3.2 --- Knockdown of Pin1 blocks ZBP-89-mediated HDAC3 reduction --- p.99 / Chapter 3.3.3 --- ZBP-89 reduces the level of IκB --- p.103 / Chapter 3.3.4 --- IκB degradation inhibitors suppresses ZBP-89-meditaed HDAC3 reduction --- p.105 / Chapter 3.3.5 --- ZBP-89 decreases HDAC3 but increases Bak in xenograft tumor tissues --- p.111 / Chapter 3.4 --- Discussion --- p.115 / Chapter Chapter Four: --- Conclusions and Future Perspectives --- p.119 / Chapter 4.1 --- Summary of results --- p.120 / Chapter 4.2 --- Conclusions --- p.121 / Chapter 4.3 --- Future Perspectives --- p.121 / References --- p.123

雖然錯誤調控微小核糖核酸 (miRNA) 引起肝細胞癌 (HCC) 發生發展的生物途徑得到了廣泛的研究，但是對於上游的調控機制卻知之甚少。以往的研究表明，組蛋白甲基化轉移酶 (EZH2) 介導的組蛋白3上27位賴氨酸三甲基化（H3K27me3）是一類通過沉默腫瘤抑制基因而誘發癌症的機制，並且與脫氧核糖核酸 (DNA) 啟動子甲基化機制獨立存在。另一方面，基因抑制也與 H3K27和DNA甲基化相關聯。盡管如此，miRNA沉默機制，特別是在肝癌中，仍然是知之甚少。 / 在這項研究中，我們使用整合全基因組定位和表達分析方法，以探討在肝癌細胞中miRNA表達的表觀遺傳和轉錄控制。通過染色質免疫沉澱偶聯人類啟動子芯片的方法，我們發現在Hep3B和HKCI - 8肝癌細胞中分別有8.4和12.2%的審問miRNA有豐富的H3K27me3。另一方面，在甲基結合域捕捉偶聯芯片實驗中，我們發現在Hep3B和HKCI-8肝癌細胞中分別有15.5和14.7% 的miRNA出現DNA超甲基化。與以往的蛋白質編碼基因結果相同，大多數 H3K27me3豐富的miRNA沒有被檢測到DNA超甲基化，並且反之亦然。 敲除EZH2基因引起H3K27me3水平廣泛下降,並且恢復 H3K27me3抑制的 miRNA表達，而DNA去甲基化劑5-氮雜 -2'-脫氧胞苷 (5-aza-dC) 卻不能重新啟動他們的表達，進一步顯示EZH2基因介導的H3K27me3引發miRNA沉默的機制是獨立存在的。然而，一些過往研究證明有腫瘤抑制功能的miRNA，包括 miR-9-1，miR-9-2和 miR-9-3 被發現同時被 H3K27me3和DNA甲基化調節。我們進一步發現，在肝瘤細胞中，miR-9 特異性調控致癌性的基因結合核因 (NF-κB) 信號通路，並且與配對的非腫瘤肝組織相比，miR-9 的表達在大約一半的原發性肝癌腫瘤（五十九分之三十零）中顯著被壓抑。 / 為了調查在H3K27me3介導的miRNA基因沉默中參與的轉錄因子，我們應用轉錄因子結合位點分析的方法檢查H3K27me3結合蛋白編碼和miRNA基因的調控區域。在包括miR-9亞型的miRNA中，滎陽 1（YY1）的結合位點在這些調控區域中反覆出現並有很高的代表性。定量芯片聯合聚合酶鏈反應結果顯示，在Hep3B細胞中，敲除YY1不僅大大降低了自身的結合力，同時在 miR-9-1，miR-9-2和 miR-9-3 的啟動子中，EZH2基因和H3K27me3結合也大大降低了。尤其重要的是，敲除YY1可以顯著地重新激活他們的表達，表明在肝癌細胞中YY1在EZH2基因介導的的miR-9 表觀遺傳沉默中發揮重要作用。功能研究證明，下調YY1能夠抑制肝癌細胞的增殖，增加細胞凋亡和減少體內的腫瘤生長。定量實時聚合酶鏈反應進一步證實在miR-9 被下調的子集肝癌腫瘤中，有超過85的樣本顯示YY1和EZH2基因同時過量表達，表明我們的研究結果具有臨床相關性。 / 總之，我們完整的分析表明miRNA的調控在肝癌上的獨特表觀遺傳模式。 H3K27me3介導的miRNA沉默可由擁有致癌功能的YY1誘發，它亦可能代表一個可能公認的肝癌癌基因。綜合表觀遺傳和miRNA表達的轉錄控制的結果，能夠提高我們對肝癌發生發展的認識和闡明利用表觀遺傳方法針對性治療肝癌的新的發展方向。 / Although the biological pathways by which mis-regulated microRNAs (miRNAs) contribute to the development of hepatocellular carcinoma (HCC) have been extensively investigated, little is known about the upstream regulatory mechanisms. Previous studies demonstrated that EZH2-mediated histone H3 lysine 27 trimethylation (H3K27me3) is a mechanism of tumor-suppressor gene silencing in cancer that is potentially independent of promoter DNA methylation. On the other hand, gene repression can be associated with both H3K27 and DNA methylation. However, the mechanisms underlying miRNA silencing, particularly in HCC, are poorly understood. / In this study, we used an integrated genome-wide location and expression analysis to investigate the epigenetic and transcriptional controls of miRNA expression in HCC cells. Chromatin immunoprecipitation (ChIP) coupled with human promoter microarrays revealed that 8.4 and 12.2% of the interrogated miRNA were enriched with H3K27me3 in Hep3B and HKCI-8 HCC cells, respectively. On the other hand, Methyl-binding domain capture coupled with microarray (MethylCap-chip) uncovered that 15.5 and 14.7% of miRNA were hypermethylated in Hep3B and HKCI-8 HCC cells, respectively. Consistent with previous observation on protein-coding genes, most of the miRNAs enriched with H3K27me3 had no detectable DNA hypermethylation and vice versa. Knockdown of EZH2 decreased global H3K27me3 level and restored expression of the H3K27me3-targeted miRNAs while the DNA demethylating agent 5-aza-2’-deoxycytidine (5-aza-dC) did not reactivate their expression, further suggesting the independence of EZH2-mediated H3K27me3 in miRNA silencing. Nevertheless, a few miRNAs reported to exhibit tumor-suppressive functions including miR-9-1, miR-9-2 and miR-9-3 were found to be regulated by both H3K27me3 and DNA methylation. We further found that miR-9 targeted the oncogenic NF-κB signaling pathway in HCC cells and were significantly repressed in approximately half of the primary HCC tumors (30/59) compared to the paired non-tumor liver tissues. / To investigate the involvement of transcription factors in H3K27me3-mediated gene silencing of miRNAs, the regulatory regions of H3K27me3-bound protein-coding and miRNA genes were submitted to transcription factor binding site analysis. The binding sites for Ying Yang 1 (YY1) were recurrently over-represented in these loci including the miR-9 isoforms. Quantitative ChIP-PCR demonstrated that knockdown of YY1 in Hep3B cells not only significantly reduced its own binding, but also the EZH2 and H3K27me3 promoter occupancy at miR-9-1, miR-9-2 and miR-9-3. Importantly, their expression levels were significantly reactivated by YY1 knockdown, suggesting that YY1 plays part in the EZH2-mediated epigenetic silencing of miR-9 in HCC cells. Functionally, down-regulation of YY1 was shown to inhibit HCC cell proliferation, increase cell apoptosis and reduce tumor growth in vivo. Quantitative RT-PCR further demonstrated that YY1 and EZH2 were concordantly over-expressed in over 85% of the same subset of HCC tumors that exhibited miR-9 down-regulation, demonstrating the clinical relevance of our findings. / In conclusion, our integrated analysis demonstrated differential epigenetic patterns of miRNA regulation in HCC. H3K27me3-mediated silencing of miRNAs may be initiated by YY1, which possesses oncogenic functions and may represent a putative HCC oncogene. The findings of combinatorial epigenetic and transcriptional control of miRNA expression enhance our understanding of hepatocarcinogenesis and shed light on the development of novel epigenetic targeted therapy of HCC. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Tsang, Pui Fong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 111-121). / Abstracts also in Chinese. / Abstract (English version) --- p.i / Abstract (Chinese version) --- p.iv / Acknowledgements --- p.vi / table of contents --- p.vii / List of tables --- p.x / List of Figures --- p.xi / Abbreviations --- p.xiii / Chapter CHAPTER 1 --- INTRODUCTION4 / Chapter 1.1 --- Hepatocellular carcinoma --- p.1 / Chapter 1.1.1 --- Epidemiology --- p.1 / Chapter 1.1.2 --- Etiology --- p.2 / Chapter 1.2 --- Epigenetic mechanisms --- p.3 / Chapter 1.2.1 --- Epigenetic silencing by DNA hypermethylation --- p.3 / Chapter 1.2.2 --- Epigenetic silencing by Polycomb group protein --- p.5 / Chapter 1.2.3 --- Interplay between H3K27me3 and DNA hypermethylation --- p.7 / Chapter 1.3 --- microRNA --- p.10 / Chapter 1.3.1 --- Transcriptional gene silencing by miRNA --- p.11 / Chapter 1.3.2 --- miRNA and cancers --- p.12 / Chapter 1.3.3 --- miRNA and liver cancer --- p.13 / Chapter 1.4 --- Signal transduction pathway and cancers --- p.14 / Chapter 1.5 --- Aims of study --- p.15 / Chapter CHAPTER 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Cell lines --- p.16 / Chapter 2.2 --- Clinical samples --- p.16 / Chapter 2.3 --- Plasmid DNA transfection --- p.16 / Chapter 2.4 --- Small interfering RNA transfection --- p.17 / Chapter 2.5 --- Extraction of total RNA --- p.19 / Chapter 2.6 --- Western blot analysis --- p.19 / Chapter 2.7 --- Quantitative RT-PCR --- p.20 / Chapter 2.8 --- miRNA Real Time RT-PCR --- p.22 / Chapter 2.9 --- ChIP-chip assay --- p.24 / Chapter 2.10 --- MethylCap-chip --- p.27 / Chapter 2.11 --- miRNA microarray --- p.28 / Chapter 2.12 --- ChIP Assay and Quantitative ChIP-PCR Assay --- p.28 / Chapter 2.13 --- Colony formation assay --- p.33 / Chapter 2.14 --- Cell proliferation assay --- p.33 / Chapter 2.15 --- Annexin V apoptosis assay --- p.34 / Chapter 2.16 --- Cancer 10-pathway reporter array --- p.34 / Chapter 2.16.1 --- Transfection of siEZH2 with 5-aza-dC treatment --- p.34 / Chapter 2.16.2 --- Transfection of siYY1 and pcDNA3-miR9 plasmid --- p.35 / Chapter 2.16.3 --- Luciferase reporter array --- p.35 / Chapter 2.17 --- Animal Studies --- p.36 / Chapter 2.18 --- Statistical Analysis --- p.36 / Chapter CHAPTER 3 --- Results / Chapter 3.1 --- Occupancy of miRNA genes by epigenetic marks in HCC cells --- p.37 / Chapter 3.1.1 --- Identification of H3K27me3-occupied miRNAs --- p.37 / Chapter 3.1.2 --- Identification of DNA methylation-occupied miRNAs --- p.41 / Chapter 3.1.3 --- Relationship between H3K27me3 and DNA methylation occupancy of miRNAs in HCC cells --- p.45 / Chapter 3.2 --- Regulation of miRNA expression by H3K27me3 and DNA methylation in HCC cells --- p.51 / Chapter 3.3 --- Epigenetic regulation and molecular function of miR-9 in HCC cells --- p.56 / Chapter 3.3.1 --- Confirmation of H3K27me3 and DNA methylation occupancy in miR-9 genes --- p.59 / Chapter 3.3.2 --- Synergistic reactivation of miR-9 upon removal of epigenetic marks --- p.62 / Chapter 3.3.3 --- Effect of miR-9 re-expression on NFKB1 (p50) expression and NF-κB signaling in HCC cells --- p.66 / Chapter 3.4 --- Role of the transcription factor YY1 in the epigenetic regulation of miR-9 --- p.72 / Chapter 3.4.1 --- Identification of transcription factors involved in the regulation of H3K27me3-bound genes --- p.72 / Chapter 3.4.2 --- Occupancy of YY1 on miR-9 in HCC cells --- p.75 / Chapter 3.4.3 --- Effects of YY1 on EZH2/H3K27me3 occupancy and expression of miR-9 --- p.78 / Chapter 3.4.4 --- Effects of YY1 on p50/p65 expression and NF-κB signaling in HCC cells --- p.81 / Chapter 3.5 --- Functional significance of YY1 in HCC --- p.84 / Chapter 3.5.1 --- Effect of YY1 on HCC cell growth --- p.84 / Chapter 3.5.2 --- Effect of YY1 on HCC cell apoptosis --- p.87 / Chapter 3.5.3 --- Effect of YY1 on HCC cell growth in vivo --- p.90 / Chapter 3.5.4 --- Expressions of YY1, EZH2 and miR-9 on clinical HCC samples --- p.92 / Chapter CHAPTER 4 --- DISCUSSION / Chapter 4.1 --- Independence of EHZ2-mediated H3K27me3 and DNA methylation --- p.97 / Chapter 4.2 --- Concordant H3K27 and DNA methylation-mediated silencing of miR-9 --- p.101 / Chapter 4.3 --- Ectopic expression of miR-9 inhibits NF-kB signaling in HCC cells --- p.102 / Chapter 4.4 --- YY1 is involved in the regulation of H3K27me3-bound genes --- p.103 / Chapter 4.5 --- Knockdown of YY1 inhibits NF-kB signaling in HCC --- p.105 / Chapter 4.6 --- Clinical relevance and therapeutic significance of miR-9 silencing by YY1-mediated recruitment of EZH2 --- p.106 / Chapter 4.7 --- Limitations and future studies --- p.109 / REFERENCES --- p.111 / PUBLICATION --- p.122

Orientador: Adriana Camargo Ferrasi / Coorientador: Maria Inês de Moura Campos Pardini / Banca: Patrícia Pintor dos Reis / Banca: Marcelo Lima Ribeiro / Resumo: O câncer gástrico é mundialmente a segunda causa de morte por câncer. No Brasil está entre os cinco neoplasias mais incidentes, mostrando-se como um grave problema de saúde pública. O conhecimento do padrão de expressão gênica de genes supressores tumorais e protooncogenes é fundamental para o esclarecimento dos mecanismos desta carcinogênese. O gene FOXE1 (forkhead box E1) pertence a uma grande família de fatores de transcrição envolvidos em processos de crescimento e diferenciação celular. Pesquisas recentes têm demonstrado o papel determinante de FOXE1 na gênese de certos tipos de neoplasias. Assim, a análise da expressão gênica de FOXE1 em amostras de tumores gástricos faz-se necessária para esclarecer seu possível envolvimento neste carcinogênese. O DNA foi extraído de 89 amostras de tumores gástricos, com seus respectivos tecidos normais adjacentes. Após o tratamento com bissulfito de sódio, o padrão de metilação foi determinado por PCR específica para metilação (MSP-PCR) com a visualização dos resultados em poliacrilamida 6% corado com nitrato de prata. 86,5% das amostras apresentaram metilação em FOXE1, sendo o mesmo padrão encontrado em tecidos normais adjacentes 74,2%. Não houve significância estatística quando os resultados foram agrupados de acordo com o sexo, tipo histológico e estadiamento. A expressão gênica foi avaliada através de qPCR para 13 dos 89 pacientes incluídos no estudo. Obteve-se para cada paciente a razão (T/N) comparando a expressão gênica da amostra de tecido tumoral (T) em relação à expressão do tecido normal adjacente ao tumor (N). 8 amostras (61,5%) apresentaram um aumento da expressão do FOXE1, com uma média de 1,6. Dentre as 5 (38,5%) amostras com QR abaixo de 1, isto é, com expressão do FOXE1 abaixo do tecido normal, a média foi de 0,7. Quanto à concordância entre o estado de metilação em FOXE1 e a sua expressão ... / Abstract: Not available / Mestre

Orientador: Ana Elizabete Silva / Coorientador: Eny Maria Goloni Bertollo / Banca: Patricia Pintor dos Reis / Banca: Cristiane Damas Gil / Resumo: Alterações nos níveis de expressão de moduladores da resposta inflamatória, como a Anexina-A1 (AnxA1/ANXA1) e a Galectina-1 (Gal-1/LGALS1) têm sido observadas em diversos tipos de câncer. O câncer colorretal, um dos modelos da associação inflamação-câncer progride, na sequência, do epitélio normal para adenoma (AD) e adenocarcinoma (ADC). Objetivos: Avaliar a expressão gênica e proteica da AnxA1 e Gal-1 e o índice de proliferação celular (IP) em amostras de AD, ADC esporádico e das mucosas normais adjacentes, assim como investigar a ocorrência de correlação entre os níveis de expressão de ambos os mRNA e de associação com fatores de risco (idade, gênero, tabagismo e etilismo) e sítio anatômico de origem da lesão. Materiais e Métodos: As análises foram realizadas pelas técnicas de PCR quantitativa em tempo real (qPCR), para quantificar os níveis de mRNA de ANXA1 e LGALS1 em 70 biópsias de lesão (27AD e 43ADC) e 58 de mucosa normal adjacente (19 e 39 respectivamente), e de imuno-histoquímica, para caracterizar a expressão proteica da AnxA1 e Gal-1 em 25 biópsias de lesão (10AD e 15ADC) e 16 de mucosa normal adjacente (6 e 10 respectivamente), como também investigar o IP pela detecção do antígeno Ki-67 em 44 biópsias de lesão (19AD e 25ADC) e 16 de mucosa normal adjacente (8 em ambas). Resultados: A expressão relativa de ANXA1 apresentou-se elevada em comparação à mucosa normal tanto no AD (RQ=1,11; P=0,040), como no ADC (RQ=2,33; P<0,001). Contudo, LGALS1 apresentou expressão relativa aumentada apenas no ADC em comparação à mucosa normal (RQ=1,85; P<0,001), enquanto no grupo AD foi observada expressão basal (RQ=0,90; P=0,319). A comparação entre as lesões mostrou que ambos os genes apresentam-se significantemente mais expressos no ADC em comparação... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Changes in expression levels of inflammatory response modulators, such as Annexin-A1 (AnxA1/ANXA1) and Galectin-1 (Gal-1/LGALS1) have been observed in several types of cancer. Colorectal cancer, one of the models of inflammation-cancer association, progresses, in sequence, from normal epithelium to adenoma (AD) and adenocarcinoma (ADC). Objectives: To evaluate the gene and protein expression of AnxA1 and Gal-1 and the cell proliferation index (IP) in samples of AD, sporadic ADC and adjacent normal mucosa, as well as to investigate the occurrence of correlation between the mRNA expression levels and association with risk factors (age, gender, smoking and drinking habits) and anatomic site of lesion origin. Materials and Methods: The analyzes were performed by quantitative real-time PCR (qPCR) technique to quantify the levels of ANXA1 and LGALS1 mRNA in 70 biopsies of lesions (27AD and 43ADC) and 58 adjacent normal mucosa (19 and 39 respectively), and immunohistochemistry technique to characterize the protein expression of AnxA1 and Gal-1 in 25 biopsies of lesions (10AD and 15ADC) and 16 adjacent normal mucosa (6 and 10 respectively), and also to investigate the IP by detection of Ki-67 antigen in 44 biopsies of lesions (19AD and 25ADC) and 16 adjacent normal mucosa (8 in both). Results: The relative expression of ANXA1 showed higher compared to adjacent normal mucosa in both AD (RQ=1.11; P=0.040) and ADC (RQ=2.33; P<0.001). However, LGALS1 mRNA showed overexpression only in ADC compared to normal mucosa (RQ=1.85; P<0.001), while in AD group it was observed basal expression (RQ=0.90; P=0.319). The comparison between lesions showed that both genes were significantly more expressed in ADC compared to AD (ANXA1: P=0.039; LGALS1: P=0.019). In both lesion groups it was observed positive correlation between the mRNA expression of these genes... (Complete abstract click electronic access below) / Mestre

Pang, Pei Shin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 96-109). / Abstract also in Chinese. / Acknowledgement --- p.i / Abstract (English) --- p.ii / Abstract (Chinese) --- p.iv / Table of Content --- p.vi / List of Figures --- p.x / List of Tables --- p.xiii / Abbreviations --- p.xiv / Chapter CHAPTER ONE: --- INTRODUCTION --- p.1 / Chapter 1.1 --- Incidence --- p.2 / Chapter 1.2 --- Etiological Factors --- p.7 / Chapter 1.2.1 --- Viral Hepatitis Infection --- p.7 / Chapter 1.2.1.1 --- Hepatitis B Virus --- p.10 / Chapter 1.2.1.2 --- Hepatitis C Virus --- p.11 / Chapter 1.2.2 --- Cirrhosis and Chronic Inflammation --- p.14 / Chapter 1.2.3 --- Dietary Aflatoxin Contamination --- p.16 / Chapter 1.2.4 --- Obesity --- p.16 / Chapter 1.3 --- Genomic Aberrations of HCC --- p.17 / Chapter 1.3.1 --- Chromosomal Imbalances --- p.17 / Chapter 1.3.2 --- Candidate Tumour Suppressor Genes and Oncogenes in HCC --- p.18 / Chapter 1.3.2.1 --- Chr 1q21-q22 gain --- p.18 / Chapter 1.3.2.2 --- Chr 8p21-p23 loss and 8q21-q24 gain --- p.18 / Chapter 1.3.2.3 --- Chr 13ql2-ql4 loss --- p.19 / Chapter 1.3.2.4 --- Chr 17pl3 loss --- p.20 / Chapter 1.3.3 --- Chromosomal Rearrangement --- p.21 / Chapter 1.4 --- Cell Lines as In-vitro Study Models --- p.22 / Chapter 1.5 --- Aim of study --- p.23 / Chapter 1.5.1 --- Objectives --- p.24 / Chapter CHAPTER TWO: --- MATERIALS AND METHODS --- p.25 / Chapter 2.1 --- Materials --- p.26 / Chapter 2.2 --- Cell Lines and Cell Culture --- p.29 / Chapter 2.2.1 --- Cell Lines --- p.29 / Chapter 2.2.2 --- Cell Culture --- p.29 / Chapter 2.3 --- Comparative Genomic Hybridization (CGH) --- p.30 / Chapter 2.4 --- Spectral Karyotyping (SKY) --- p.31 / Chapter 2.5 --- Expression Profiling --- p.34 / Chapter 2.6 --- Functional Investigations --- p.37 / Chapter 2.6.1 --- Growth Kinetics --- p.37 / Chapter 2.6.2 --- Cytotoxic Assay --- p.38 / Chapter CHAPTER THREE: --- RESULTS --- p.39 / Chapter 3.1 --- Molecular Cytogenetic Analysis --- p.40 / Chapter 3.1.1 --- Comparative Genomic Hybridization (CGH) --- p.40 / Chapter 3.1.1.1 --- Introduction --- p.40 / Chapter 3.1.1.2 --- CGH Results --- p.41 / Chapter 3.1.1.3 --- Clustering Analysis of CGH Data --- p.49 / Chapter 3.1.2 --- Spectral Karyotyping (SKY) --- p.51 / Chapter 3.1.2.1 --- Introduction --- p.51 / Chapter 3.1.2.2 --- Ploidy Status --- p.51 / Chapter 3.1.2.3 --- Structural Rearrangements --- p.54 / Chapter 3.1.2.4 --- Chromosomes Susceptible to Further Rearrangements --- p.63 / Chapter 3.1.2.5 --- Maintained Common HCC Translocations --- p.65 / Chapter 3.2 --- Expression Profiling --- p.67 / Chapter 3.2.1 --- Introduction --- p.67 / Chapter 3.2.2 --- Gene Expression Profiling --- p.69 / Chapter 3.2.3 --- The Ontologies of Deregulated Genes --- p.71 / Chapter 3.2.4 --- Maintained Biological Pathways --- p.74 / Chapter 3.3 --- Functional Investigation --- p.76 / Chapter 3.3.1 --- Introduction --- p.76 / Chapter 3.3.2 --- Cell Morphology --- p.76 / Chapter 3.3.3 --- Growth Kinetics --- p.79 / Chapter 3.3.4 --- Cytotoxic Assay --- p.83 / Chapter CHAPTER FOUR: --- DISCUSSION --- p.86 / Chapter 4.1 --- Introduction --- p.87 / Chapter 4.2 --- Molecular Cytogenetic Analysis --- p.87 / Chapter 4.3 --- Expression Profiling --- p.91 / Chapter 4.4 --- Conclusion --- p.94 / REFERENCES --- p.95