子宮内膜癌是最常见的妇科恶性肿瘤，其中有80%属于子宮内膜腺样癌，这一癌症发病的分子机制尚未清楚。研究表明，癌基因表达异常或者功能异常在肿瘤的发生发展过程中具有重要的作用。另一方面，抑癌基因在肿瘤细胞中特异性甲基化失活通常导致细胞恶性转变和肿瘤的发生。本实验将阐明癌基因阴阳1和抑癌基因PCDH10在人类子宮内膜腺样癌发病中的作用。 / 本实验第一部分研究多功能转录因子阴阳1（YY1）在子宮内膜腺样癌发病过程中的作用。首先本实验证实YY1在子宮内膜腺样癌临床标本和癌细胞系中均明显表达上调，并且上调的程度与肿瘤的FIGO分期相关。接着体外细胞培养和裸鼠荷瘤模型的实验均提示抑制YY1 表达可抑制癌细胞增殖和体外迁移，而过表达YY1则促进癌细胞增殖。这些结果表明YY1在子宮内膜腺样癌发病中具有促进作用。进一步全细胞基因组转录谱分析提示YY1 介入子宮内膜腺样发病的各个方面，并通过抑制抑癌基因APC的表达发挥发挥重要作用。深入的分子机制研究发现一个新的表观抑制作用模型：YY1可募集EZH2等多梳蛋白到APC启动子区并导致后者组蛋白3赖氨酸27上三甲基化，从而抑制APC基因转录。此外，本实验还发现YY1在子宮内膜腺样癌的表达增高是由于微小RNA，miR-193a-5p，在此癌中表达下降所导致的。所以，本实验第一部分的结果揭示了miR-193a-5p-YY1-APC这条全新的信号通路在子宮内膜腺样癌发病中发挥重要作用，并可作为潜在的治疗靶点。 / 本实验第二部分鉴定出PCDH10作为子宮内膜腺样癌一个新的抑癌基因。通过5-氮杂-2'-氧胞嘧啶处理和亚硫酸氢钠测序的方法，我们证实抑癌基因PCDH10在子宮内膜腺样癌中失活是由于其启动子区DNA甲基化所致，并且这种DNA甲基化介导的PCDH10表达沉默在子宮内膜腺样癌临床标本和癌细胞系中很常见，但不存在于正常子宮内膜组织。另外，在子宮内膜腺样癌细胞系体外实验中恢复PCDH10的表达可抑制细胞增殖、单细胞克隆形成，促进细胞凋亡。 同时在体实验荷瘤模型中恢复PCDH10的表达也可抑制肿瘤细胞增殖，这些结果与其肿瘤抑制功能相符。 / 总之，本实验结果阐明了YY1和PCDH10在子宮内膜腺样癌发病过程中新的作用，拓展了子宮内膜腺样癌发病分子机制的研究并为其药物治疗提供了潜在的靶点。 / Endometrial cancer is the most common gynecologic malignancy and about 80% of these cancers are endometrial Endometrioid carcinoma (EEC). The molecular mechanisms underlying EEC tumorigenesis are under-explored. Aberrant expression and function of oncogenes promote tumor development by modulating many aspects of tumor cell growth. On the other hand, tumor specific promoter methylation on tumor suppressor genes (TSG), which are generally unmethylated in normal cells, usually initiate and promote malignant transformation and cancer initiation. Our study aims to characterize the functions of an oncogenic transcription factor Yin Yang 1 (YY1) and a novel tumor suppressor gene PCDH10 in Human Endometrioid Endometrial Adenocarcinoma. / In the first part of our study, we investigated the function of a multifunctional TF, YY1 in EEC tumorigenesis. We demonstrated YY1 is up-regulated in EEC cell lines and primary tumors and its expression is associated with FIGO stages. Depletion of YY1 inhibits EEC cell proliferation and migration both in vitro and in vivo whereas over-expression of YY1 promotes EEC cell growth. These results suggest that YY1 functions as an onocogenic factor in EEC. Transcriptome analysis revealed a significant effect of YY1 on critical aspects of EEC tumorigenesis and its down-regulation of APC transcripts. Further mechanistic investigation uncovered a new epigenetic silencing mode of Adenomatosis Polyposis Coli (APC) by YY1 through recruitment of EZH2 and trimethylation of histone 3 lysine 27 in its promoter region. Additionally, YY1 over-expression was found to be a consequence of miR-193a-5p down-regulation through direct miR-193a-5p-YY1 interplay. Our results therefore established a novel miR-193a-5p-YY1-APC regulatory axis contributing to EEC development, which may serve as future intervention target. / In the second part of our study, we identified PCDH10 as a novel tumor suppressor gene in EEC. By using bisulfate genomic sequencing combined with pharmacologic demethylation drug treatment, we elucidated that PCDH10 inactivation in EEC is a consequence of DNA hypermethylation on its promoter region. Further study suggested that hypermethylation-mediated PCDH10 silencing was a common event in EEC cell lines and clinical samples, but not in normal endometrial tissues. Restoration of PCDH10 expression in EEC cells suppressed cell proliferation, inhibited single cell colony formation and induced cell apoptosis; moreover, overexpression of PCDH10 inhibited EEC xenograft tumor growth in vivo.These results suggest PCDH10 acts as a tumor suppressor. / Together, our results reveal the novel functions of YY1 and PCDH10 in EEC. These findings add novel insights into the molecular mechanisms of EEC development and progression, which may serve as potential therapeutic targets for this disease. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Yang, Yihua. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 170-186). / Abstracts also in Chinese. / TITLE --- p.I / ABSTRACT --- p.III / ACKNOWLEDGEMENTS --- p.VII / PUBLICATION --- p.IX / ABBREVIATIONS --- p.X / LIST OF FIGURES --- p.XIII / LIST OF TABLES --- p.XVI / TABLES OF CONTENT --- p.XVII / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Endometrioid Endometrial Adenocarcinoma (EEC) --- p.1 / Chapter 1.1.1 --- Epidemiology --- p.2 / Chapter 1.1.2 --- Etiology and risk factors --- p.3 / Chapter 1.1.3 --- Treatment and prognosis --- p.8 / Chapter 1.1.4 --- Molecular Mechanisms --- p.9 / Chapter 1.1.5 --- APC and Wnt/β-catenin signaling pathway --- p.12 / Chapter 1.1.6 --- Summary --- p.18 / Chapter 1.2 --- Epigenetic modifications and EEC --- p.20 / Chapter 1.2.1 --- Epigenetic modifications --- p.20 / Chapter 1.2.2 --- Epigenetic and cancer --- p.21 / Chapter 1.2.5 --- Summary --- p.30 / Chapter Chapter 2 --- Material and Method --- p.31 / Chapter 2.1 --- Tissue samples --- p.31 / Chapter 2.2 --- Cell culture --- p.32 / Chapter 2.3 --- Cell proliferation assays --- p.33 / Chapter 2.4 --- Cell migration assay --- p.34 / Chapter 2.5 --- 3-deazaneplanocin A (Dznep) or 5-aza-2'-deoxycytidine (5-aza) treatment --- p.34 / Chapter 2.6 --- Computational prediction --- p.35 / Chapter 2.7 --- Cell cycle assay --- p.35 / Chapter 2.8 --- Apoptosis assay --- p.36 / Chapter 2.9 --- Total RNAs, Total proteins and Genomic DNA extraction --- p.36 / Chapter 2.10 --- Bisulfite Genomic Sequencing --- p.38 / Chapter 2.11 --- Oligonucleotides --- p.39 / Chapter 2.12 --- RT-PCR, Semi-quantitative PCR and Real-time PCR --- p.41 / Chapter 2.13 --- microRNA validation --- p.43 / Chapter 2.14 --- Plasmid construction --- p.43 / Chapter 2.15 --- Transfection --- p.45 / Chapter 2.16 --- Luciferase reporter assay --- p.45 / Chapter 2.17 --- Western blotting --- p.46 / Chapter 2.18 --- Immunofluorescence ( IF ) --- p.48 / Chapter 2.19 --- Immunohistochemistry (IHC) --- p.50 / Chapter 2.20 --- ChIP assay --- p.53 / Chapter 2.21 --- Sequencing and base calling --- p.55 / Chapter 2.22 --- Read mapping to genome with splice-aware aligner sequenced --- p.55 / Chapter 2.23 --- Xenograft mouse model --- p.55 / Chapter 2.24 --- Statistical analysis --- p.57 / Chapter Chapter 3 --- Yin Yang 1 Plays an Oncogenic Role in Human Endometrioid Endometrial Adenocarcinoma --- p.58 / Chapter 3.1 --- YIN YANG 1(YY1) --- p.58 / Chapter 3.1.1 --- YY1 structure --- p.58 / Chapter 3.1.2 --- YY1 function --- p.59 / Chapter 3.1.3 --- YY1 and epigenetic --- p.61 / Chapter 3.1.4 --- YY1 and cancer --- p.62 / Chapter 3.1.5 --- Regulation of YY1 expression and activity --- p.66 / Chapter 3.2 --- Results --- p.68 / Chapter 3.2.1 --- YY1 is up-regulated in EEC lines and localizes in nuclei of EEC cells --- p.68 / Chapter 3.2.2 --- YY1 expression level is associated with EEC clinicopathological features --- p.72 / Chapter 3.2.3 --- Knock-down of YY1 by RNAi inhibits EEC cell proliferation and migration --- p.77 / Chapter 3.2.4 --- Ectopic expression of YY1 promotes EEC cell proliferation --- p.84 / Chapter 3.2.5 --- YY1 does not affect EEC cell cycle and cell apoptosis --- p.91 / Chapter 3.2.6 --- Genome-wide characterization of YY1-mediated transcriptome changes --- p.94 / Chapter 3.2.7 --- Gene Ontology analysis of YY1 targets on EEC tumorigenesis --- p.98 / Chapter 3.2.8 --- YY1 inhibits APC gene expression and functions --- p.101 / Chapter 3.2.9 --- YY1 inhibits APC expression through recruiting EZH2 and causing H3K27me3. --- p.105 / Chapter 3.2.10 --- Knock-down of YY1 does not change DNA methylation status of CpG island of APC gene --- p.117 / Chapter 3.2.11 --- SiYY1 oligo injection inhibits tumor grows in vivo --- p.119 / Chapter 3.2.12 --- miR-193a-5p is down-regulated in EEC cell lines and clinical samples --- p.126 / Chapter 3.2.13 --- miR-193a-5p targets YY1 3’UTR and inhibits YY1 expression --- p.128 / Chapter 3.2.14 --- miR-193a-5p inhibits tumor grow in vivo --- p.133 / Chapter 3.3 --- Discussion --- p.136 / Chapter 3.3.1 --- YY1 oncogenic functions in EEC --- p.136 / Chapter 3.3.2 --- YY1 epigenetically silences APC --- p.138 / Chapter 3.3.3 --- miR-193a-5p down-regulates YY1 in EEC --- p.139 / Chapter 3.4 --- Conclusion --- p.141 / Chapter Chapter 4 --- The tumor suppressive functions of PCDH10 in Human Endometrioid Endometrial Adenocarcinoma --- p.143 / Chapter 4.1 --- Introduction --- p.143 / Chapter 4.1.1 --- PCDH10 structure and function --- p.143 / Chapter 4.1.2 --- PCDH10 and tumor --- p.146 / Chapter 4.1 --- Results --- p.149 / Chapter 4.2.1 --- PCDH10 is down-regulated in EEC cell lines and clinical samples --- p.149 / Chapter 4.2.2 --- PCDH10 is hypermethylated in EEC cell lines and clinical samples --- p.150 / Chapter 4.2.3 --- Pharmacologic demethylation restores PCDH10 expression in EEC cell lines --- p.152 / Chapter 4.2.4 --- Ectopic over-expression of PCDH10 inhibits EEC cell proliferation --- p.154 / Chapter 4.2.5 --- PCDH10 over-expression induces EEC cell apoptosis --- p.161 / Chapter 4.2.6 --- PCDH10 over-expression inhibits tumor grows in vivo --- p.166 / Chapter 4.3 --- Discussion and future plan --- p.169 / REFERENCE --- p.170
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328438 |
| Date | January 2012 |
| Contributors | Yang, Yihua, Chinese University of Hong Kong Graduate School. Division of Obstetrics and Gynaecology. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | electronic resource, electronic resource, remote, 1 online resource (xx, 186 leaves) : ill. (some col.) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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