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研究生:林子軒
研究生(外文):Tzu-Hsuan Lin
論文名稱:Snail調節肝癌細胞中MMP9和ZEB1基因表達的轉錄機制
論文名稱(外文):The transcriptional mechanism for Snail to mediate gene expression of MMP9 and ZEB1 in hepatoma cell
指導教授:吳文陞
指導教授(外文):Wen-sheng Wu
學位類別:碩士
校院名稱:慈濟大學
系所名稱:醫學檢驗生物技術學系醫學生物技術碩士班
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:65
中文關鍵詞:基因調節癌轉移
外文關鍵詞:SnailEMTmetastasis
相關次數:
  • 被引用被引用:1
  • 點閱點閱:577
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
鋅指轉錄因子的成員Snail可能引發上皮間質轉化(EMT)在胚胎發育和腫瘤轉移所需。很多基因的轉錄都已經知道會被Snail正向或負向調控。在轉錄抑制中,Snail可以連接E-box上共識的序列,從而干擾上皮細胞標誌蛋白,如E - cadherin的基因表達。
  然而,迄今Snail如何上調基因表達的機轉還不明確。我們最近的研究顯示,在人類肝癌HepG2細胞中,12 - O- tetradecanoylphorbol-13 - acetate(TPA)誘導Snail和EGR – 1及SP- 1 共同作用上調p15INK4b的轉錄。同時也發現在p15INK4b啟動子上,有一個可能的Snail結合位TCACA,剛好位於EGR-1/SP-1重疊區域的上游。
  我們進一步研究一個可被Snail上調的基因:基質降解酶,基質金屬蛋白酶9(MMP9)的轉錄機制。使用deletion mapping分析,我們確定TPA-responsive element,位於MMP-9啟動子的轉錄起始位點上游-950〜-771 bp. 有趣的是,這一區域還包含上面提到的Snail 結合 位,而且也位於EGR-1/SP-1重疊區域的上游。如果Snail和EGR-1/SP-1結合區域被突變,TPA誘導 MMP9啟動子的活性就會降低。
  此外,染色質免疫沉澱分析法進一步證明了TPA誘導Snail , EGR- 1和SP- 1,結合在他們各自的啟動子區域。目前,我們還研究了其他可被TPA誘導且經由Snail上調的基因,包括ZEB-1的轉錄機制。初步結果表明,在這些基因的啟動子上的TPA反應位置,也包含上面提到的Snail結合位,並位於EGR-1/SP-1重疊區域的上游。
總而言之,我們的結果強調,Snail與 EGR-1/SP-1合作並結合到共同的啟動子位置而上調基因表達。
The Snail families of zinc-finger transcription factors may trigger epithelial mesenchymal transition (EMT) required for embryonic development and tumor metastasis. A lot of genes were known to be either positively or negatively regulated by Snail. As a transcriptional repressor, Snail may bind to consensus sequence E-box, thus interfering gene expression of epithelial markers such as E-cadherin.
  However, how Snail upregulate gene expression is not clear thus far. Our recent report demonstrated Snail associates with EGR-1 and SP-1 to upregulate transcription of p15INK4b induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in human hepatoma HepG2 cells. We found a novel sequence motif TCACA upstream of EGR-1/SP-1 overlapping region for Snail to bind on p15INK4b promoter.
  We further investigated transcriptional mechanism of matrix metalloproteinase 9 (MMP9), one of the matrix degradation enzyme which is also upregulated by Snail. Using deletion mapping analysis, we identify TPA-responsive element of MMP-9 promoter located between -950~-771 bp upstream of the transcriptional initiation site. Interestingly, this region also contain proposed Snail-target site upstream of EGR-1/SP-1 overlapping region. TPA –induced MMP9 promoter activity abolished if the binding regions of Snail and EGR-1/SP-1 were mutated.
  Also, ChIP assay further demonstrated TPA induced binding of Snail, EGR-1and SP-1 to their respective promoter region. Currently, we also investigate transcriptional mechanisms of other TPA-inducible, Snail-upregulated genes including ZEB-1. The preliminary results demonstrated TPA-responsive elements within promoters of these genes also contain proposed Snail-target site upstream of EGR-1/SP-1 overlapping region.
Collectively, our result highlighted that Snail cooperate with EGR-1/SP-1 to upregulate gene expression via binding to concensus promoter region.
目錄
Abstract 6
中文摘要 8
[1]研究背景 9
[2]研究動機與目的 11
[3]研究材料與方法 12
1.Cell culture and chemicals 12
2. RNA purification 12
3. Reverse transcription polymerase chain reaction(RT-PCR) 12
4. MMP9 and ZEB1 promoter plasmid construction 13
5.Transient transfection 17
6.Small hairpin RNA 17
7.Luciferase assay and Dual luciferase assay 18
8.Western blotting assay 19
9.Chromatin immunoprecipitation (ChIP) assay 21
[4]研究結果 25
1.TPA誘導MMP9啟動子不同區域所造成的活性 25
2.TPA誘導MMP9啟動子主要區域所造成的活性 26
3.TPA誘導Snail之基因表現 27
4.Snail是TPA調控MMP9-pro950啟動子之轉錄活化所需要 27
5.TPA誘導Hep G2細胞株中ZEB1的表現 28
6.TPA誘導ZEB1啟動子不同區域所造成的活性 29
7.TPA誘導ZEB1啟動子主要區域所造成的活性 30
8.In vivo探討受TPA調控之轉錄因子與MMP-9啟動子結合之情形 30
9.In vivo探討受TPA調控之轉錄因子與ZEB1啟動子結合之情形 31
[5]討論與未來研究方向 32
[6]參考文獻 35
[7]圖表目錄 38
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