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研究生:吳啟生
研究生(外文):Chi Sheng Wu
論文名稱:利用差異性甲基化雜交法分析並研究GRIK2基因在胃癌中扮演的角色
論文名稱(外文):Global Analysis of Methylated Genes in Gastric Cancer by Differential Methylation Hybridization and CpG DNA Microarray and Identification of Glutamate receptor, ionotropic, kainate 2 as a Potential Tumor Suppressor Gene in Gastric Cancer
指導教授:張玉生張玉生引用關係
指導教授(外文):Y. S. Chang
學位類別:博士
校院名稱:長庚大學
系所名稱:生物醫學研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2009
畢業學年度:98
論文頁數:96
中文關鍵詞:甲基化、胃癌
外文關鍵詞:Differential Methylation Hybridization、CpG DNA Microarray、GRIK2、Tumor Suppressor Gene、Gastric Cancer
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中文摘要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2007年排名全國癌症死亡第七位。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,遺傳因子,飲食,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。其中,在染色體變異因子中,腫瘤抑制基因啟動子區域的高度甲基化,是引起癌症發生的重要因子之ㄧ。因此,在本研究中,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的胃部組織進行比對,篩選出一系列受到甲基化調控的基因。並針對其中之 glutamate receptor, ionotropic, kainate 2 (GRIK2)基因啟動子區域甲基化現象作進一步分析,同時確定該區域在胃癌細胞及組織中為高度甲基化。GRIK2的甲基化,可以明確區分臨床檢體中腫瘤與非腫瘤區域;而且,GRIK2基因甲基化與基因表現程度,經同時分析27對檢體,發現在同一對檢體中,呈現負相關性(r=-0.44)。進一步探討GRIK2與癌症生成之相關性,發現表現GRIK2的AGS及AZ521胃癌細胞株,在「附著性依賴實驗」中,細胞群落的數目明顯少於控制組;同時表現GRIK2之細胞株,表現出明顯抑制細胞的移動能力,這些證據足以說明GRIK2在胃癌中,可能具有抑制腫瘤移動的能力,進而達到扮演抑制腫瘤形成的特性。至於GRIK2基因起動子甲基化,作為胃癌診斷之生物指標,未來仍需要更多的驗證工作。
Abstract Aberrant DNA methylation is considered as a major mechanism for silencing tumor suppressor genes in gastric cancer. Here, we used CpG microarray and differential methylation hybridization (DMH) strategies to identify potential tumor suppressor genes, and recovered glutamate receptor, ionotropic, kainate 2 (GRIK2) as a novel epigenetic target in gastric cancer. Additional experiments revealed that the promoter region of GRIK2 was hypermethylated in three out of the four tested gastric cancer cell lines, and its expression was restored by treatment of cells with the DNA methylation inhibitor, 5’-aza-dC. In clinical samples, the GRIK2 promoter was differentially hypermethylated in 27 paired tumor tissues compared to adjacent normal tissues (P<0.001), and this methylation was inversely correlated with the expression level of GRIK2 mRNA (r=-0.44). Functional studies further revealed that GRIK2-expressing gastric cancer cell lines showed decreased colony formation and cell migration. Taken together, these results suggest that GRIK2 may play a tumor suppressor role in gastric cancer. Future studies are warranted to examine whether DNA hypermethylation of the GRIK2 promoter can be used as a potential tumor marker for gastric cancer.
ContentIntroduction 1 I. Epigenetics 1 A. DNA methylation 1 B. DNA methylation and cancer 2 C. DNA methyltransferases (DNMTs) 3 1. DNMT1 4 2. DNMT2 4 3. DNMT3A and DNMT3B 4 D. Histone proteins modification 5 II. Gastric cancer 6 A. Epidemiology 6 B. Risk of gastric cancer 7 1. Diet 7 2. Cigarette and alcohol 7 3. Helicobacter pylori infection 7 C. Biological markers of gastric cancer 8 III. Glutamate receptors 9 A. Metabotropic glutamate receptor 10 B. Ionotropic glutamate receptors 11 1. AMPA receptor 11 2. NMDA receptor 12 3. Kainate receptor 13 4. Glutamate receptor, ionotropic, kainate 2 (GluR6 or GRIK2) 13 Specific aims 15 Material and methods 16 Preparation of CpG microarray 16 Differential Methylation Hybridization (DMH) and CpG microarray 16 Cell culture, clinical samples and 5’-aza-dC treatment 18 Bisulfite treatment and bisulfite sequencing 19 Quantitative real-time RT-PCR 19 RNA extraction, cDNA synthesis and quantitative real-time RT-PCR 20 Transfection and immunofluorescence 20 Establishment of stable transfectants 21 Immunohistochemical staining analysis 21 Western blot analysis 22 Anchorage-independent colony formation assay 22 Cell proliferation assay 23 Wound closure assay 23 Transwell migration assay 23 Statistical analysis 24 Result 25 Differential methylation methylation and CpG microarray data analysis 25 Methylation status of deleted in liver cancer 1 (DLC-1) gene in gastric cancer 25 Potential candidate tumor suppressor genes of gastric cancer 26 Hypermethylation of glutamate receptor, ionotropic kanate 2 (GRIK2) CpG islands in gastric cancer cell lines 27 Hypermethylation of the GRIK2 promoter region in clinical samples 28 GRIK2 does not alter cell proliferation but does affect anchorage-independent colony formation…in AGS and AZ521 cells 31 GRIK2 suppress cell migration in AGS and AZ521 cells 31 GRIK2 does not affect cell migration ability and colony formation ability in KATO III cell lines 32 -catenin colocolizes with GRIK2 on cell membrane but does not involve in cell migration mechanism of gastric cancer cell lines 33 Discussion 35 References 42 Table 52 Figure and Appendix 60
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