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系統識別號 U0026-0812200913510389
論文名稱(中文) 第一型去氧核醣核酸甲基轉移酵素的表現受缺氧調控之探討
論文名稱(英文) Regulation of DNA methyltransferase 1 under hypoxia
校院名稱 成功大學
系所名稱(中) 生理學研究所
系所名稱(英) Department of Physiology
學年度 95
學期 2
出版年 96
研究生(中文) 吳軍緯
研究生(英文) Chun-wei Wu
電子信箱 s3693101@mail.ncku.edu.tw
學號 s3693101
學位類別 碩士
語文別 英文
論文頁數 102頁
口試委員 指導教授-蔡少正
召集委員-孫孝芳
口試委員-呂昱瑋
中文關鍵字 外基因  缺氧誘導因子  甲基化  缺氧 
英文關鍵字 DNA methylation  epigenetics  hypoxia  hypoxia-inducible factor 
學科別分類
中文摘要 去氧核醣核酸甲基化現象是由去氧核醣核酸甲基轉移酵素所造成,為一種可抑制基因轉錄作用之外基因修飾。不正常的去氧核醣核酸甲基化現象與去氧核醣核酸甲基轉移酵素的表現量高度相關,且可能導致腫瘤相關基因的表現失調以及癌症的惡化;然而,目前去氧核醣核酸甲基轉移酵素基因之轉錄表現的調控機制尚未完全了解。在腫瘤形成的過程中,細胞常常面臨缺氧的困境。細胞可藉由活化缺氧誘導因子來調控許多缺氧相關基因的表現,使得細胞得以適應缺氧環境並維持氧氣的供需平衡。我們發現缺氧處理可以抑制第一型去氧核醣核酸甲基轉移酵素基因的訊息核醣核酸及蛋白質的表現,並降低內生性啟動子的轉錄活性;利用化學藥劑,如鐵離子螯合劑(desferrioxamine; DFO)或是二甲基酸草醯基甘胺酸(dimethyloxaloylglycine; DMOG),處理細胞以造成缺氧誘導因子在有氧環境下累積後,也可觀察到相同的結果。利用核醣核酸干擾技術則證實,缺氧藉由誘發第一型與第二型缺氧誘導因子以抑制第一型去氧核醣核酸甲基轉移酵素基因的訊息核醣核酸之表現。此外,我們在第一型去氧核醣核酸甲基轉移酵素基因啟動子的上游,發現一個高度保留的缺氧反應組序列,具有促進子之功能並可與第一型缺氧誘導因子結合。外生的第一型去氧核醣核酸甲基轉移酵素基因的啟動子可受缺氧刺激而誘發其活性;其中位於啟動子上游的缺氧反應組,以及位於5’端未轉譯區域的兩個5’-GCGTG-3’序列則參與在此正向調控中。內生與外生的第一型去氧核醣核酸甲基轉移酵素基因啟動子對缺氧刺激的反應完全相反,我們懷疑可能還有其他的因子在缺氧的刺激下內生的啟動子轉錄活性。總的來說,缺氧刺激可以透過缺氧誘導因子造成第一型去氧核醣核酸甲基轉移酵素基因的表現量下降。藉此,缺氧刺激可能得以影響基因組上的去氧核醣核酸甲基化狀態而全面性地調控基因表現。
英文摘要 DNA methylation, which is mediated by DNA methyltransferases (DNMTs), acts as an epigenetic modification to regulate genes expression including oncogenes and tumor suppressor genes. Aberrant DNA methylation, which is highly correlated with expression level of DNMTs, may contribute to malignancy of tumor cells owing to dysregulation of many tumor-related genes. However, the regulation of DNMTs expression is not fully understood. Hypoxic stress has been shown to involve in several biological processes including tumorigenesis. Hypoxia inducible factor (HIF) is the major transcription factor, which can regulate expression of hypoxia-response genes under hypoxic conditions, and promotes adaptation of cells to hypoxia. In study presented, we found that hypoxia suppresses expression of DNMT1 mRNA and protein, and DNMT1 transcription rate in vivo. The down-regulation was mirrored by desferrioxamine (DFO) and dimethyloxaloylglycine (DMOG)-mimic hypoxia highlighting the role of HIF in hypoxia-mediated DNMT1 suppression. We also identified a highly conserved hypoxia-responsive element (HRE) located at -385/-368 in human DNMT1 promoter region. This HRE matrix enhanced SV-40 promoter activity and was bound by HIF-1 in vitro under hypoxia. Unexpectedly, hypoxia-induced exogenous DNMT1 promoter activity was mediated by HRE matrix (-385/-368) and two GCGTG boxes within 5’ un-translated region. This observation was contrary to the down-regulation of endogenous DNMT1 promoter activity by hypoxia; hence, we assumed other factors may regulate DNMT1 promoter in vivo under hypoxia. For the first time, we proved that hypoxia suppresses expression of DNMT1. By regulating DNMT1 expression, hypoxia may regulate genome-wide gene expression via controlling DNA methylation, a well-known epigenetic change observed in many physiological and pathological processes.
論文目次 Abstract in chinese i
Abstract ii
Acknoledgments iii
Table of Content iv
List of Figures vi
List of Tables vii
Introduction 1
Materials and Methods 12
Cell culture and treatments 12
Isolation of RNA 13
Real-time RT-PCR 14
Isolation of genomic DNA 15
Isolation of total protein 16
Isolation of nuclear protein 17
Lowry assay 17
Western blot 18
Plasmids construction 18
Minipreparation of plasmid DNA 20
Transfection and promoter-activity assay 21
Electrophoretic mobility shift assay (EMSA) 22
SiRNA 23
Statistical analysis 24
Results 25
Hypoxia suppressed the expression of DNMT1 in HeLa cell 25
Chemical-mimic hypoxia inhibited the expression of DNMT1 26
Hypoxia suppressed DNMT1 expression in cancer and normal cells 26
Hypoxia suppressed the expression of DNMT1 through HIF 27
Hypoxia down-regulated expression of DNMT1 is controlled at transcriptional level 28
Functional validation of highly conserved HRE matrix in human DNMT1 promoter 28
Hypoxia induced promoter activities in cells transiently transfected with full-length human DNMT1 promoter constructs 30
cis-elements in 5’ un-translated region mediated the activation of exogenous human DNMT1 promoter activity by hypoxia 32
The expression of DNMT1 mRNA was activated by 5-aza-dC 33
Discussion 34
References 43
Appendix 86
I. Reagents and buffers used in cell culture 86
II. Reagents and buffers used in RNA isolation 87
III. Reagents and buffers used in DNA electrophoresis 87
IV. Reagents and buffers used in total/nuclear protein isolation and western blot 88
V. Antibodies used in the study 90
VI. Reagents and buffers used in Lowry assay 90
VII. Reagents and buffers used in preparation of plasmid DNA 91
VIII. Reagents and buffers used in promoter assay 91
IX. Reagents and buffers used in EMSA 92
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