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系統識別號 U0026-0307201416080800
論文名稱(中文) 研究在SW620細胞中WWP2 isoforms之間的相互調控及對訊息傳遞的影響
論文名稱(英文) WWP2 isoforms differentially modulate the signaling pathway in SW620 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 102
學期 2
出版年 103
研究生(中文) 黎乃萱
研究生(英文) Nai-Hsuan Li
學號 S26014023
學位類別 碩士
語文別 英文
論文頁數 44頁
口試委員 指導教授-呂增宏
口試委員-馬明琪
口試委員-劉校生
中文關鍵字 WWP2  E3 ubiquitin ligase  SW620 cells 
英文關鍵字 WWP2  E3 ubiquitin ligase  SW620 cells 
學科別分類
中文摘要 WWP2 (WW domain-containing protein 2)屬於NEDD4-like E3 ubiquitin-protein ligase的一員。過去已有研究發現其能夠對PTEN進行泛素化 (ubiquitination)的作用,並且也有研究發現WWP2能將phosphatidyl inositol 3-kinase (PI3K)活化。雖然有許多研究發現WWP2對於癌細胞的生長具有一定程度的影響,而人類大腸直腸癌細胞SW620本身具有很高的WWP2表達,但真正的作用機轉仍然未知。Eps8 (Epidermal growth factor receptor kinase substrate 8)為EGFR以及Src的受質之一,會參與在由EGFR所調控的作用機轉之中,像是細胞的增生作用。WWP2有三個isoforms,分別是full-length WWP2 (WWP2-FL 或V1), C-terminal isoform (WWP2-C 或V2),以及 N-terminal isoform (WWP2-N或V3)。已有研究發現WWP2 isoforms可以調控TGFβ/Smads 的訊息傳遞,進而影響epithelial mesenchymal transition (EMT)的產生。然而,WWP2的三個isoforms在活化Eps8/Src 及PI3K的訊息傳遞路徑上扮演的角色還並不清楚。為了要釐清WWP2 isoforms對於細胞生長的影響,我們利用SW620細胞創造出同時將WWP2-V1/V2 knockdown (G type)以及同時將WWP2-V1/V3 knockdown (H type)的細胞。 在siWWP2-H knockdown的細胞中,我們看到AKT, AKT pi- S473, Src, Src pi-Y416, FAK, FAK pi-Y861以及K-Ras的表達量下降,而細胞生長也變緩慢,表示WWP2-V1或WWP2-V3或兩者同時影響著細胞生長的速度。為了更深入探討是哪個isoform造成的影響,我們將V1 resistance construct 放回到siWWP2-H knockdown的HeLa細胞中。此時我們看到比起HeLa細胞本身以及siWWP2-H knockdown的HeLa細胞,放回V1 resistance construct的細胞生長變得更緩慢。由以上結果我們認為WWP2 isoforms,尤其是WWP2-V3,參與在Src/FAK 和PI3K/AKT 的訊息傳遞路徑上扮演著相當重要的角色。
英文摘要 WWP2 (WW domain-containing protein 2) is a NEDD4-like E3 ubiquitin-protein ligase whose activity has been indicated in the ubiquitination of PTEN and elevating the activity of phosphatidyl inositol 3-kinase (PI3K). Although it has been indicated in cell proliferation in cancer cells, the underlying molecular mechanism is still vague. Eps8 (Epidermal growth factor receptor kinase substrate 8) functions in the EGFR-mediated cell proliferation and is a common substrate of both EGFR and Src. Colorectal cancer cells SW620 have a significant amount of WWP2 expression whereas Eps8 affects cell growth. There are three WWP2 isoforms, which are full-length WWP2 (WWP2-FL or V1), C-terminal isoform (WWP2-C or V2), and N-terminal isoform (WWP2-N or V3). WWP2 isoforms have previously been shown to regulate TGFβ/Smads signaling activity linked to epithelial mesenchymal transition (EMT). However, the distinct role of the three WWP2 isoforms involving in the activation of Eps8/Src and PI3K and cell proliferation is still unknown. To clarify the effect of the three WWP2 isoforms on cell proliferation, we generated siWWP2-G knockdown cells, which targets WWP2-V1 and WWP2-V2, and siWWP2-H knockdown cells, which targets WWP2-V1 and WWP2-V3. Decreased levels of AKT, AKT pi- S473, Src, Src pi-Y416, FAK, FAK pi-Y861, K-Ras and cell proliferation/migration were observed in siWWP2-H knockdown cells, indicating WWP2-V1 or WWP2-V3 or both of them plays an important role in cell proliferation. To clarify the distinct effect of WWP2-V1 and WWP2-V3, we transfected V1 resistance construct into siWWP2-H type HeLa knockdown cells. Interestingly, cell proliferation was further decreased when V1 resistance construct was transfected into siWWP2-H HeLa knockdown cells while no significant difference on cell proliferation was shown in vector-transfected siWWP2-H HeLa cells. Taken together, our results highlight the importance of WWP2 isoforms, particularly WWP2-V3, involving in the Src/FAK and PI3K/AKT pathway-mediated cell proliferation.
論文目次 Abstract 2
中文摘要(Abstract in Chinese) 3
Acknowledgement 5
Table of Contents 6
Abbreviations 7
Introduction 9
Materials and Methods 15
Results 23
Discussion 27
References 30
Figures 35
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