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研究生:謝文定
研究生(外文):Wen-Ting Hsieh
論文名稱:從附基因調控層次上探討TSA處理後人類乳癌細胞MCF7中c-fos及c-jun基因表現差異之機制
論文名稱(外文):Characterization of Human c-fos and c-jun Gene Expression and Epignetic Regulation in TSA treated MCF7
指導教授:徐明達徐明達引用關係
指導教授(外文):Ming-Ta Hsu
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生化暨分子生物研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:66
中文關鍵詞:附基因調控乳癌細胞甲基化組蛋白修飾
外文關鍵詞:MCF7c-fosepigeneticmethylation
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從附基因調控的層次上探討基因的表現與否,主要是探討DNA甲基化和組蛋白的修飾兩個層面上。根據研究已知人類乳癌細胞株MCF-7在加入TSA之後,會導致MCF-7的細胞週期停止在G0/G1期以及造成細胞凋亡。c-fos和c-jun這兩個基因是立即性早期反應基因,會形成AP-1複合體,AP-1複合體是重要的轉錄因子,調控下游許多和細胞生長及細胞凋亡有關基因的表現。依據affymatrix的資料顯示,MCF-7在處理TSA 24小時之後,c-fos和c-jun都有大量表現的情形發生。TSA本身是抗癌藥,同時也是HDACi (histone deacetylase inhibitor),一個會改變附基因調控機制的藥物。所以我們利用bisulfite定序方式研究TSA處理24小時之後,c-fos和c-jun整個基因以及包括基因上游promoter的位置DNA甲基化程度上的改變並且利用ChIP研究c-fos和c-jun在組蛋白修飾上的變化。根據結果顯示處理TSA 24小時之後,c-jun在DNA甲基化改變的程度上變化不大,c-fos在處理TSA之後,位在基因上游2000個鹼基對以及第一個intron的區域變得較為去甲基化。根據ChIP的結果顯示,c-fos和c-jun兩者在TSA處理過後H3K9乙醯基化的情形不管是在位置或是程度上確實都增加了。綜合研究結果指出TSA可能主要是影響c-fos在第一個intron位置的DNA去甲基化、H3K9去雙甲基化和增加乙醯基化,打開原本轉錄停止的位置,使其表現量大增。而TSA則是會去影響c-jun的CpG island shore區域的附基因調控,使H3K9去雙甲基化和增加乙醯基化,而這樣的改變造成c-jun大量表現主要是在組蛋白的修飾層次,而非DNA甲基化的層次。
The study of gene expression based on epigenetic regulation level mainly includes DNA methylation and histone modifications. According to previous studies, the cell cycle of MCF-7 becomes arrested in G0/G1 phase and apoptosis is induced after TSA treatment for 24 hours. C-fos and c-jun are immediate early genes that form the AP-1 complex. The AP-1 complex is a very important transcription factor that regulates many downstream genes including genes that regulate cell growth and apoptosis. Based on affymetrix microarray data, c-fos and c-jun were upregulated after 24 hours treatment of TSA in MCF-7. TSA is an anti-cancer drug and also a HDACi (histone deacetylase inhibitor) drug that alters epigenetic mechanism. I used bisulfite sequencing to study the DNA methylation pattern of the whole gene and the promoter region of c-fos and c-jun after TSA treatment. I also used ChIP to study the histone modifications of c-fos and c-jun. I found that the DNA methylation pattern of c-jun showed similar patterns before and after TSA treatment. However, the upstream 2000 bps and intron 1 of c-fos becomes unmethylated after TSA treatment. According to ChIP analysis, the level of H3K9 acetylation of c-fos and c-jun increases after TSA treatment. Finally, the results revealed that TSA may affect the demethylation of first intron of c-fos DNA, decrease H3K9 dimethylation and increase H3K9 acetylation to relieve the premature termination block site in intron 1 and upregulate c-fos expression. TSA treatment resulted in the decreased H3K9 dimethylation and increased H3K9 acetylation at the CpG island shore of c-jun to upregulate c-jun expression without affecting the DNA methylation of the gene. The epigenetic regulation mechanism of this gene therefore is at histone modification level but not at DNA methylation level.
中文摘要----------------------------------------------1
英文摘要----------------------------------------------2
背景--------------------------------------------------4
材料和方法-------------------------------------------12
實驗結果---------------------------------------------20
討論-------------------------------------------------25
附圖-------------------------------------------------30
附錄-------------------------------------------------49
參考文獻---------------------------------------------55
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