Epigenetic and Post-transcriptional Regulation of Cancer-associated Genes in the Avian Ovarian Carcinomas

학위논문 (석사)-- 서울대학교 대학원 : 농생명공학부(바이오모듈레이션전공), 2013. 2. 송권화.Ovarian Cancer is the most lethal gynecological disease among women due to difficult to diagnose at early stage and lack of molecular biomarkers. To overcome this limitations and obstacles, research is required to gain a better understanding of ovarian carcinogenesis mechanisms both genetics and epigenetics in avian species, especially laying hens which are the best animal model for research on human ovarian carcinogenesis. The aims of this study with laying hens were to determine: 1) the expression of DNA methyltransferases (DNMTs), cyclins, cyclin dependent kinases and their inhibitors, and pleiotrophin (PTN) gene in normal and cancerous ovaries2) whether these genes are regulated by post-transcriptional actions of specific microRNAsand 3) methylation patterns of several specific genes. DNMTs are key regulators of DNA methylation and have crucial roles in carcinogenesis, embryogenesis and epigenetic modification. Results of the present study demonstrated increased expression of DNMT genes in cancerous ovaries of laying hens and post-transcriptional regulation of those genes by specific microRNAs, as well as control of hypermethylation of the promoters of tumor suppressor genes. The cell cycle system is controlled in a timely manner by three groups of cyclins, cyclin dependent kinases and their inhibitors. Abnormal alterations of cell cycle regulatory mechanisms are a common feature of many diseases including numerous tumor types such as ovarian cancer. Results of the present study demonstrated increased expression of cell cycle-related genes in cancerous ovaries of laying hens and indicate that expression of these genes is post-transcriptionally regulated by specific microRNAs. PTN is a developmentally-regulated growth factor which is widely distributed in various tissues and also detected in many kinds of carcinomas. Our results indicated that PTN is most abundant in the GE of adenocarcinoma of cancerous, but not normal ovaries of hens. Bisulfite sequencing revealed that 30- and 40% of -1311 and -1339 CpG sites are demethylated in ovarian cancer cells, respectively. Further, several microRNAs, specifically miR-499 and miR-1709 were discovered to influence PTN expression via its 3′-UTR which suggests that post-transcriptional regulation influences PTN expression in chickens. Collectively, this present study provides new insights into epigenetic regulation and functional roles in ovarian carcinogenesis in laying hens that are highly relevant to the development of therapies for treatment of ovarian cancers in humans. As well as, it absolutely provides better approach to identify epithelial derived human ovarian cancer.CONTENTS SUMMARY i CONTENTS iii LIST OF FIGURES v LIST OF TABLES vii LIST OF ABBREVIATONS viii CHAPTER 1. GENERAL INTRODUCTION 1 CHAPTER 2. LITERATURE REVIEW 5 1. Characteristics of Ovarian Cancer 6 1.1. General characteristics 6 1.2. Classification 7 2. Genetic and Epigenetic Mechanisms of Cancerous Development 8 2.1. Genetic Mechanisms 8 2.2. Epigenetic Mechanisms 9 3. Animal Models for Human Ovarian Cancer Research 11 3.1. Primate Models 11 3.2. Rodent Models 11 3.3. Avian Models 12 4. Cell Cycle Regulatory Genes in Ovarian Cancer 13 CHAPTER 3. EPIGENETIC FUNCTIONS AND DNA METHYLATRNASFERASES IN THE CHICKEN OVARIAN CARCINOMAS 15 1. Abstract 16 2. Introduction 18 3. Materials and Methods 20 4. Results 30 5. Discussion 40 CHAPTER 4. DISTINCT EXPRESSION PATTERN AND POST-TRANSCRIPTIONAL REGULATION OF CELL CYCLE GENES IN THE GLANDULAR EPITHELIA OF AVIAN OVARIAN CARCINOMAS 44 1. Abstract 45 2. Introduction 47 3. Materials and Methods 49 4. Results 57 5. Discussion 68 CHAPTER 5. CHICKEN PLEIOTROPHIN GENE: IDENTIFICATION AND REGULATIONS OF TISSUE SPECIFIC EXPRESSION BY ESTROGEN IN THE OVIDUCT AND DISTINCT EXPRESSION PATTERN IN THE GLANDULAR EPITHELIA OF OVARIAN CARCINOMAS 73 1. Abstract 74 2. Introduction 75 3. Materials and Methods 79 4. Results 88 5. Discussion 105 CHAPTER 6. GENERAL DISCUSSION 111 REFERENCES 115 SUMMARY IN KOREAN 142 ACKNOELEDGEMENTS 146Maste