臺灣博碩士論文加值系統

胃癌主要好發於開發中與已開發國家。以台灣地區為例，胃癌致死率在2008年排名全國癌症死亡第五位。雖然胃癌引起的死亡率在全球各地顯示出逐年下降的趨勢，但是發生初期徵兆並不明顯；目前之方法檢測出胃癌病變時，多已進入中期甚至末期。因此，對於胃癌之檢測與治療，依舊是現階段處理胃癌非常重要的課題。環境因子，飲食，抽菸，遺傳因子，染色體變異以及幽門螺旋桿菌感染都是引起胃癌發生的主要因子。此外，上遺傳因子亦可調控腫瘤抑制基因啟動子區域的高度甲基化，為引起癌症發生的重要機制之ㄧ。本研究即以建立DNA甲基化分析平台以便於尋找DNA高度甲基化之抑癌基因。我們利用快速篩選平台 Differential Methylation Hybridization以及自備的Human CpG DNA microarray，由胃癌細胞株與正常胃部組織作比對，篩選出107個受到甲基化調控的基因，並針對其中之 Low density lipoprotein receptor-related protein 1B (LRP1B) 基因啟動子區域甲基化現象作進一步分析。結果顯示，該區域在四個不同胃癌細胞珠中均為高度甲基化，且在四個細胞珠中，LRP1B 甲基化程度與其表現程度成負相關。此外，利用甲基化抑制劑處理細胞，可恢復 LRP1B 表現。在74個胃癌腫瘤組織中，45個 (60.8%) 具有高度甲基化的情形。且診測19個胃癌腫瘤組織，其中15個LRP1B 表現量在腫瘤組織中明顯低於腫瘤旁正常組織。進一步探討LRP1B與癌症生成之相關性，大量表現 LRP1B於胃癌細胞株AGS顯示LRP1B具有抑制細胞生長之能力、 減少細胞群落數目、並且具有抑制腫瘤在老鼠體內生成的能力。這些證據足以說明LRP1B可能為胃癌之腫瘤抑制基因，並與胃癌之形成相關。

Gastric cancer is the second most common cancer worldwide, while in Taiwan it is the fifth leading cause of cancer-related death. Several factors have been implicated in the development of gastric cancer, including chromosomal instability, genetic and epigenetic alterations, Helicobacter pylori infection, and environmental factors such as diet and smoking. Among them, the aberrant methylation of CpG dinucleotides has been reported during tumorigenesis in gastric cancer. In this study, we used CpG island microarray and differential methylation hybridization (DMH) to identify novel epigenetic targets in gastric cancer. We identified 107 genes that were differentially hypermethylated in the four gastric cancer cell lines (AGS, AZ521, KATOIII and TMC1) but not in the two adult normal stomach tissue samples. Several tumor suppressor genes previously identified as being regulated by DNA methylation in gastric cancer were identified by our approach. One of the candidate genes, LRP1B, was hypermethylated in four gastric cancer cell lines and clinical samples (45 out of 74, 60.8%), and low LRP1B mRNA expression was associated with high methylation levels in gastric cancer cell lines. Addition of DNA methylation inhibitor (5-Aza-dC) can restore the mRNA expression of LRP1B in these cell lines, indicating that DNA methylation is involved in regulating LRP1B expression. In 15 out of 19 (78.9%) gastric tumor tissues, LRP1B mRNA expression was significantly lower than in adjacent normal tissues. In addition, ectopic expression of mLRP1B4 in gastric cancer cell lines suppressed cell growth, colony formation and tumor formation in nude mice. These results collectively indicate that LRP1B is a functional tumor suppressor gene in gastric cancer, and that is regulated by DNA methylation.

中文摘要 1
Abstract 2
Introduction 3
I. Epigenetics modification 3
A. DNA methylation 3
B. Histone modification 5
C. RNA associated silencing 6
D. DNA methylation mechanism for gene silencing 7
E. DNA methylation and cancer 7
F. Detection of epigenetic modifications 9
II. Gastric cancer 10
A. Epidemiology 10
B. Risk factors 11
Environmental factors 11
Helicobacter pylori infection 12
Genome instability 12
Epigenetic alterations 13
III. Low density lipoprotein (LDL) receptor 14
IV. Low density lipoprotein receptor-related protein 1B (LRP1B) 17
Specific aims 19
Material and methods 20
Cell lines and tissue samples 20
CpG microarray 20
Differential methylation hybridization (DMH) 21
Bisulfite treatment and sequencing 21
Quantitative methylation-specific PCR (Q-MSP) 22
5-Aza-2’-deoxycytidine (5-Aza-dC) treatment and real-time Q-RT-PCR analysis 22
Quantitative genomic PCR 23
Transfection and selection of stable cell lines 23
Western blot analysis 23
Anchorage-dependent colony formation assay 24
Anchorage-independent colony formation assay 24
Growth curve 24
Flow cytometry 24
Tumorigenicity in nude mice 25
Wound closure assay 25
Invasion assay 26
Statistical analysis 26
Results 27
Preparation of the CGI microarray 27
Differential methylation hybridization (DMH) and data analysis 28
Confirmation of microarray findings 28
LRP1B hypermethylation in gastric cancer clinical samples 31
Identification of deletions of LRP1B by quantification real-time genomic PCR 32
Restoration of LRP1B expression in gastric cancer cell lines following treatment with 5-Aza-dC 33
LRP1B mRNA expression in paired gastric cancer tissues 33
Ectopic expression of LRP1B suppresses colony formation, cell growth, and tumor formation in nude mice 34
LRP1B suppresses cell growth in gastric cancer cell line by prolonging the G2/M phase in cell cycle 35
LRP1B did not affect cell migration and invasion 36
Discussion 37
References 44
Figures 55
Tables 79
Abbreviations 89
Appendix 91

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