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研究生:劉琪玲
研究生(外文):Chi-Ling Liu
論文名稱:表觀遺傳修飾作用與甘胺酸氮甲基轉移酵素基因表現之相關性研究
論文名稱(外文):A Study of The Regulation of Epigenetic Modification Associated With GNMT Gene Expression
指導教授:陳宜民陳宜民引用關係
指導教授(外文):Ying-Ming A. Chen
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:86
中文關鍵詞:甘胺酸氮甲基轉移&甘胺酸氮甲基轉移&甘胺酸氮甲基轉移&甘胺酸氮甲基轉移&甘胺酸氮甲基轉移&甘胺酸氮甲基轉移&甘胺酸氮甲基轉移&
外文關鍵詞:Epigenetic ModificationMethylationHistoneGNMTMicroarray
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表觀遺傳調控(epigenetic regulation)調控染色質的機制經由非基因體序列中的資訊來主導,其中包含了DNA甲基化(DNA methylation) 及組蛋白修飾(Histone modification)。DNA及組蛋白甲基化共同調控染色質構造及基因表現,並且這些甲基化的異常被發現和癌症以及多種疾病有關。甘胺酸氮甲基轉移酵素(Glycine-N-methyltransferase;GNMT)是細胞內維持S-adenosylmethionine (SAM)與S-adenosylhomocysteine (SAH)平衡的重要酵素,在人類肝癌細胞株及組織的表現量會被抑制,而在GNMT基因剃除鼠中亦被發現會自發性產生肝癌,因此GNMT為一肝癌腫瘤抑制基因。本研究利用CGH及CpG Island甲基化晶片技術全面性偵測GNMT剔除鼠與野生鼠基因體甲基化程度差異,並且將甲基化狀態與基因表現情況做整合。由CGH晶片結果顯示GNMT剔除鼠呈現全面性低度甲基化的現象,在CpG island晶片分析中發現GNMT剔除鼠甲基化有改變的基因大部分參與在MAPK及Wnt訊號傳遞路徑中,而且有性別差異,在公鼠中甲基化改變區域皆達顯著差異。在In vivo試驗中發現,利用realtime PCR方式發現GNMT剔除鼠參與組蛋白甲基化的相關酵素SET7表現量上升,LSD1、Suv39H1、Suv39H2及JMJD3表現量下降,並且利用西方墨點法觀察肝組織中與其相對應的組蛋白甲基化現象也有改變;H3K27me3於母鼠中有顯著上升,公鼠則有下降趨勢。H3K4me2無顯著差異。H3K9me2在公鼠中有下降趨勢,H3K9me3在母鼠中有增加趨勢。在In vitro試驗中則發現將肝癌細胞株HepG2之GNMT抑制之後, SET7表現量上升,LSD1表現量下降,與其相對應的組蛋白甲基化H3K4me2增加,H3K9me2、 H3K9me3下降。此研究結果顯示GNMT剔除之後會造成甲基化變異,顯示可能經由表觀遺傳調控機制以及影響細胞訊息傳遞路徑來導致癌化的發生。
Epigenetic modifications play important roles in diverse biological processes ranging from regulation of gene expression, embryonic development to human diseases such as cancers. Histone methylation and DNA methylation cooperatively regulate chromatin structure and gene activity. Glycine N-methyltransferase (GNMT) is a vital metabolic enzyme that regulates one-carbon metabolism. Previously, we reported that the expression of GNMT was down-regulated in HCC cell lines and tumor tissues, and that the GNMT-/- mice developed HCC spontaneously. Therefore, GNMT is a tumor suppressor for liver cancer. Genome-wide methylation patterns in GNMT-/- mice and wild type mice were investigated by using CGH arrays and CpG island arrays, and correlated with expression arrays. The CGH arrays analysis showed that the global DNA hypomethylation in GNMT-/- mice. The CpG island array and pathway analysis showed that the methylation pattern was changed mostly in genes involved in MAPK and Wnt signaling pathway. These changes were gender difference and more significant in males. Real-time PCR was used to exam the expression levels of enzymes responded to histone methylation. The results showed that SET7 was increased and LSD1, Suv39H1, Suv39H2 and JMJD3 were decreased in GNMT-/- mice. The results of western blot showed that the patterns of total histone methylation were changed in GNMT-/- mice liver tissues. H3K27me3 were significantly increased in females, but were decreased in males. H3K9me2 were decreased in males and H3K9me3 were increased in females. There was no statistic significant difference in H3K4me2. In vitro, the expression level of SET7 was increased and LSD1 was decreased when GNMT was knocked down in HepG2 cells. In addition, total H3K4me2 were increased, and H3K9me2 and H3K9me3 were decreased. Therefore, GNMT deficiency could result in methylation patterns changed in genes and histones and alter the cell signaling, especially in MAPK and Wnt signaling, that would be involved in the tumorigenesis of HCC.
中文摘要 i
Abstract ii
目錄 iv
圖目錄 vi
表目錄 viii
第一章 研究背景 1
一. 肝癌與甲基化 1
二. 表觀遺傳修飾:甲基化修飾作用 2
三. 甘胺酸氮甲基轉移酵素 4
四. 晶片式比較型基因體雜交分析技術(Array-CGH) 6
五. CpG island 晶片 7
第二章 研究目的 8
第三章 材料與方法 9
第四章 結果 23
一. 比較型基因體雜交甲基化晶片(Comparative Genomic Hybridization microarray;CGH array)分析 23
二. CpG island 甲基化晶片分析 23
三. 甲基化基因群驗證 24
四. 整合與比較甲基化晶片(Methylation array) 與基因表現晶片(expression array)資料 25
五. 利用in vitro系統偵測細胞株內GNMT表現量與整體基因組甲基化之相關性 26
六. 組蛋白甲基轉移酵素(methyltransferases)與去甲基酵素(dymethylases)表現量分析 26
七. GNMT表現量與組蛋白甲基化修飾之相關性分析 27
第五章 討論 29
一. 探討GNMT對於DNA甲基化之影響 29
二. 探討甲基化晶片與基因表現晶片之間關聯性 31
三. 探討GNMT對於組蛋白甲基化之影響 31
四. 探討GNMT於基因表觀遺傳調控之影響 34
第六章 結論及未來方向 35
第七章 參考文獻 36
圖表 40
附錄 65

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