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研究生:柯智鐘
研究生(外文):Chih-Chung Ko
論文名稱:GCIP在胃癌細胞轉移之角色
論文名稱(外文):The role of GCIP in metastatic gastric cancer cells
指導教授:莊秀美莊秀美引用關係
口試委員:林芸薇闕斌如
口試日期:2011-07-02
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物醫學研究所
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:2011
畢業學年度:99
語文別:英文
論文頁數:31
中文關鍵詞:胃癌轉移訊號傳遞
外文關鍵詞:GCIPgastric cancer cellmetastasissignal transduction
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GCIP在先前的研究中證實是一個共同抑制因子,並參與在細胞生長、發育和分化當中。在最近的研究中更發現,GCIP蛋白能抑制癌細胞的生成及生長,但是,GCIP蛋白在抑制癌細胞的生成及生長之分子機制調控仍然是未知的。 本篇研究利用胃癌細胞為研究對象,我們發現活化態的ERK1/2和p38蛋白量與GCIP的蛋白質的表現量剛好相反。當處理MEK1/2和p38的抑制劑後,我們發現ERK和p38訊號路徑可以調控GCIP蛋白表現量。進一步利用免疫沉澱及分析蛋白上連接的泛素量,發現ERK和p38會與GCIP有交互作用,並且透過泛素連接水解系統促進GCIP的水解。更進一步研究發現, GCIP第328位置的酥胺酸(Threonine)可以被ERK或p38接上磷酸根後促進其水解。利用細胞轉移試驗,我們發現GCIP抑制癌細胞的轉移能力。綜合以上結果,我們發現ERK和p38 MAPK訊號可以將GCIP磷酸化後促進GCIP的水解。此外,GCIP會促使胃癌細胞的轉移能力下降。

GCIP functions as a potential negative regulator of transcription, and involves in the cell growth, development, differentiation. Recent studies have indicated that GCIP functions as the suppressor for tumorigenesis and tumor growth. However, the mechanism of GCIP in the suppression of tumorigenesis and cancer cell growth is still unknown. In this study, we used gastric cancer cell lines as the model and found that the levels of phosphorylated ERK1/2 and p38α were opposite to GCIP protein levels in AGS and MKN-45 cells. ERK1/2 or p38 MAPK inhibitor experiments showed that GCIP protein expression was down-regulated by ERK1/2 and p38 kinase signaling pathways. Furthermore, immunoprecipotation followed by analyzing ubiquitylation on GCIP showed that ERK1/2 and p38 could interact with GCIP and then promoted GCIP degradation by ubiquitin-proteasome pathway. Moreover, substitution of T328 with alanine of GCIP attenuated GCIP degradation and decreased the phosphor-Thr-Pro signaling, indicating that phosphorylation on T328 might play a role on GCIP ubiquitination and degradation. Furthermore, migration assay showed that GCIP could repress cancer cell migration. Taken together, these results indicate that ERK and p38 MAPK can phosphorylate GCIP to promote GCIP protein degradation through ubiquitin-proteasome pathway. In addition, we show that GCIP represses the migration ability of gastric cancer cells.

中文摘要..................................................i
Abstract…………………………………………………………………..ii
Chapter1. Introduction
1.1 Cellular function of GCIP................................................................1
1.2 GCIP in cancer……………………………………....…………….2
1.3 MAPK pathway……………………………………….……………4
1.4 Metastasis…………………………………....…………………….6
Chapter2. Aim…………………………………………………...…………………..7
Chapter3. Material and method
3.1. Antibodies and chemicals……………………………………........8
3.2 Cell lines, and cell culture………………………………………....8
3.3 Cell lysate preparation, immunoprecipitation and immunoblotting..........................................................................................9
3.4 Inhibitors treatment with cells………………….…………….…….9
3.5 GCIP cloned and site-direct mutagenesis…………………….…...9
3.6 Transfection and RNA interference……………………...…….…10
3.7 Reverse transcriptase-polymerase chain reaction (RT-PCR)..................................................................................................10
3.8 Cell migration assay.......................................................................11
Chapter4. Result
4.1 Distinct expression of GCIP in different cancer cell lines...........................................................................................................13
4.2 ERK1/2 and p38 MAPK decreased GCIP protein expressionthrough 26S proteasome degradation............................................13
4.3 ERK and P38 MAPK interacted with GCIP and facilitated GCIP degradation through phosphorating GCIP....................................14
4.4 GCIP repressed cancer cell migration...........................................16
Chapter5. Discussion................................................................................................17
Chapter6. References................................................................................................18
Chapter7. Figures...................................................................................20

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