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系統識別號 U0026-0608201214510100
論文名稱(中文) 研究在HeLa細胞中WWP2參與在Eps8促進細胞增生作用的角色
論文名稱(英文) Studying the role of WWP2 in Eps8-promoted cell proliferation in HeLa cells
校院名稱 成功大學
系所名稱(中) 藥理學研究所
系所名稱(英) Department of Pharmacology
學年度 100
學期 2
出版年 101
研究生(中文) 童郁琇
研究生(英文) Yu-Hsiu Tung
學號 s26994053
學位類別 碩士
語文別 中文
論文頁數 71頁
口試委員 指導教授-呂增宏
口試委員-馬明琪
口試委員-洪建中
口試委員-洪良宜
中文關鍵字 Eps8  WWP2  細胞增生  HeLa cell 
英文關鍵字 Eps8  WWP2  cell proliferation  HeLa cell 
學科別分類
中文摘要 泛素化(ubiquitination)是蛋白質後轉譯修飾之一,它能調控許多生理功能。WWP2是屬於HECT-type E3 ligase一員,最近的研究中已發現許多HECT-type E3s和癌症的進程有關。而WWP2在癌細胞扮演的角色卻是較少被探討的。已知在多種人類癌症的病人中,Eps8 (EGF receptor pathway substrate No.8) 這個致癌因子被發現有過度表現的情形。而且Eps8也是致癌因子EGFR和Src的受質。從實驗室之前的研究知道,在大腸直腸癌細胞SW620中發現WWP2能和Eps8產生交互作用並且能被ubiquitination。因此我們便想了解WWP2和Eps8的表現與交互作用對於癌細胞生長有何影響?從我們實驗發現,在HeLa細胞中暫時性表達WWP2會減少FAK、Pi-S473 AKT和CyclinD1的表達量,而且也影響細胞增生(cell proliferation)和colony的形成。然而在H618-4 (Eps8-attenuated HeLa)細胞中暫時性表達WWP2則看到FAK Pi-Y861、Src Pi-Y416和AKT Pi-S473的表達上升以及增加cell proliferation和colony生長的情形。此結果在穩定表達WWP2的H618-4細胞(H618-4/V1-5)中也可發現。在暫時性表達WWP2的HeLa細胞中以RT-PCR分析FAK的mRNA並沒有改變,顯示WWP2調控FAK表達是在post-transcriptional level上。然而因表達WWP2所造成FAK減少並非是透過proteosome所調控。最後,在大腸直腸癌SW620細胞中將WWP2 knockdown後,也會造成FAK Pi-Y861、Src Pi-Y416、AKT Pi-Y473減少以及抑制cell proliferation。由以上結果我們認為WWP2在不同細胞內可經由Src/FAK 和 PI3K/AKT傳遞路徑影響細胞生長。
英文摘要 Protein ubiquitination is an important post-translational modification that regulates numerous biological functions. Although the HECT type E3 ubiquitin ligases had been correlated with cancer development, the role of WWP2 in cancer formation remains to be solved. EPS8 (EGF receptor pathway substrate No.8) is a common substrate of EGF receptor and Src. Here, we demonstrated that EPS8 associated with WWP2 and was ubiquitinated in SW620 cellls. We wondered whether WWP2 affected EPS8 expression and participated in cell growth. Our data indicated that transient overexpression of WWP2 does not affect Eps8 expression but inhibits Pi-S473 AKT, CyclinD1 expression, and decreased cell proliferation in both culture dishes and soft agar in HeLa cells. In contrast, transient overexpression of WWP2 in Eps8-attenuated HeLa cells elevates FAK Pi-Y861, AKT Pi-S473, and Src Pi-Y416 resulted in the increase of cell proliferation. Similar results were observed in stably WWP2-overexpressing Eps8-attenuated cells. Further study indicated that WWP2 overexpression did not affect FAK mRNA level in HeLa cells. Since treatment with MG132 could not restore FAK expression in these cells, WWP2-mediated FAK expression probably was not through proteosome-dependent pathway. Finally, we demonstrated that attenuation of WWP2 deceased FAK Pi-Y861, Src Pi-Y416, AKT Pi-Y473 expression and suppressed cell proliferation in SW620 cells. Taken together, our results indicated that WWP2 may affect the cell growth by Src/FAK and PI3K/AKT pathways depending on the cellular context.
論文目次 中文摘要 1
英文摘要 4
目錄 9
縮寫簡表 10
第一章 緒論 13
第二章 實驗材料與方法 23
第一節 實驗材料 24
第二節 實驗方法 27
第三章 實驗結果 41
第四章 討論 48
第五章 圖表 54
參考文獻 66
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