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系統識別號 U0026-0812200914104406
論文名稱(中文) Eps8促進人類子宮頸癌細胞生長、移行與化療抗藥性之探討及其對預後之影響
論文名稱(英文) Eps8 promotes proliferation, migration and drug resistance of human cervical cancer cells, and its effect on patient survival
校院名稱 成功大學
系所名稱(中) 基礎醫學研究所
系所名稱(英) Institute of Basic Medical Sciences
學年度 96
學期 2
出版年 97
研究生(中文) 陳韻如
研究生(英文) Yun-Ju Chen
電子信箱 s5891120@mail.ncku.edu.tw
學號 S5891120
學位類別 博士
語文別 中文
論文頁數 84頁
口試委員 口試委員-洪文俊
口試委員-馬明琪
口試委員-沈孟儒
口試委員-李政昌
指導教授-呂增宏
召集委員-賴明德
中文關鍵字 Eps8 
英文關鍵字 Eps8 
學科別分類
中文摘要 致癌蛋白Eps8可以促進大腸癌細胞之增生與移行。為了探究Eps8在人類子宮頸癌中所扮演的角色,我們由45位屬於早期子宮頸癌的患者檢體切入,以免疫組織染色的方式檢視Eps8的表現。與周圍正常的子宮頸上皮組織相比,Eps8的表現在癌化組織中有增加的情形,此現象亦在in vitro細胞培養的條件下得到證實。進一步Kaplan-Meier分析法得到的結果顯示Eps8表現較高的病患檢體,患者的預後較差。這個結果顯示Eps8在子宮頸癌中有促進惡化的作用,因此我們便以siRNA的方式,進行一系列的研究。Eps8表現減少的HeLa與SiHa子宮頸癌細胞,無論是在in vitro細胞增生或in vivo腫瘤生長上,都比控制組細胞來得慢。細胞週期的分析結果顯示Eps8表現降低會造成G1-phase arrest,而Eps8減弱導致的p53蛋白囤積,p21Waf 1/Cip 1表現上升,cyclins D1,D3,E的表現下降及Rb hyperphosphorylation的減少也解釋了G1-phase arrest的形成原因。值得注意的是,Eps8減弱導致的p53蛋白囤積,至少部份是由於p53蛋白半衰期增長所致。同時,我們也證實了Eps8有促進子宮頸癌細胞化療抗藥性的作用,而p53蛋白堆積的增加及Src與AKT活性的下降可能是造成Eps8減弱所致之抗藥性降低的原因。而將Eps8送回Eps8表現減低之HeLa細胞則可使上述這些因Eps8減弱所產生的變化(包括對於細胞週期、腫瘤生長及化療敏感度的作用)有一定程度的回復,顯示eps8 siRNA的作用是具有專一性的。在細胞移行方面,Eps8減弱會降低HeLa及SiHa細胞的移行能力,FAK活性及表現,MMP-9表現及basal ERK活性,而在送回Eps8的HeLa細胞中則發現這些變化有回復的情形。同時,以PD98059抑制ERK活化可抑制HeLa及SiHa細胞中MMP-9的表現。進一步以PD98059處理,可以在程度上有差別的降低HeLa,Eps8減弱及回送Eps8之HeLa細胞的移行能力。以專一性的MMP-9抑制劑處理也可觀察到同樣的情形。綜合以上結果,我們在子宮頸癌細胞中確立了Eps8可經由活化ERK而促進MMP-9的表現;同時,此條訊息傳遞路徑參與在部份Eps8所調控的子宮頸癌細胞移行能力中。最後,本論文的這些新發現讓我們對Eps8在人類子宮頸癌上所扮演的病理角色有較深入的了解。
英文摘要 The oncoprotein Eps8 promotes colon cancer proliferation and migration. In an attempt to elucidate the roles of Eps8 in human cervical cancer, we began the study by examining immunohistochemically Eps8 expression in specimens of 45 early-stage cervical cancer patients. Eps8 expression was increased in cervical carcinoma compared to neighboring normal cervical epithelia. In vitro studies also indicated that Eps8 expression was upregulated in cervical carcinoma cells. Kaplan-Meier analysis of these 45 patients demonstrated an inverse relationship between the level of Eps8 protein expression and percentage of patient survival. By virtue of small interference RNA (siRNA) methodology, reduced in vitro proliferation and in vivo tumor formation were observed in Eps8-attenuated HeLa and SiHa cervical cancer cells. Cell cycle analysis demonstrated that Eps8 attenuation resulted in G1-phase arrest, which might be attributed to increased p53 protein accumulation and p21Waf1/Cip1 protein expression, reduced cyclins D1, D3, E expression and a concomitant decrease in Rb hyperphosphorylation. It was worth noting that decreased p53 protein turnover rate contributed to increased p53 protein accumulation resulted from Eps8 attenuation. Furthermore, we demonstrated for the first time that increased Eps8 expression conferred better chemoresistance on cervical cancer cells. Reduced chemoresistance in Eps8-attenuated HeLa and SiHa cells might be attributed to increased p53 protein accumulation and decreased Src and AKT activity. Ectopic Eps8 expression in Eps8-attenuated HeLa cells could reverse the aforementioned biological events, indicating that the effect of eps8 siRNA was specific. With respect to cellular mobility, Eps8 attenuation decreased motility, FAK activity and expression in cervical cancer cells. MMP-9 expression and basal ERK activity were also reduced in Eps8-attenuated cells. Restored Eps8 expression could recover MMP-9 expression, FAK activity and expression, and migratory ability in these cells. Suppression of ERK activity by PD98059 in cervical cancer cells repressed MMP-9 expression. Furthermore, PD98059 and MMP-9 specific inhibitor could decrease mobility differentially in HeLa and its derived cells. Taken together, we established a signaling cascade in which Eps8 could enhance MMP-9 expression by promoting ERK activation, and this cascade contributed to Eps8-mediated cervical cancer cell mobility. These new findings deepen our understanding towards the pathological roles of Eps8 in human cervical cancer.
論文目次 縮寫檢索表
中文摘要 …………………………………………………... 3
英文摘要 …………………………………………………... 5

第一章 緒論
I. 致癌蛋白Eps8 ( Eps8 as an oncoprotein ) …………… 7
II. 子宮頸癌 (Cervical Cancer) ………………………… 8
III. 腫瘤抑制基因p53與Rb ………………………....... 9
IV. MMP-9與細胞移行 ………………………………… 11
V. 本論文研究目標 …………………………………… 13

第二章 實驗材料及方法
第一節 實驗材料 ……………………………………… 14
第二節 實驗方法 ……………………………………… 17

第三章 實驗結果
1. 人類子宮頸癌Eps8的表現量影響罹癌患者之預後 ……… 32
2-1. Eps 8表現量的減低會抑制體外及體內子宮頸癌細胞
之生長速度 .................................................................. 33
2-2. Eps 8對於細胞週期的影響 ................................................... 33
2-3. Eps8對於cell cycle modulators的調控 …………………… 34
3. Eps8表現量的降低會增進子宮頸癌細胞對cisplatin與
paclitaxel兩藥物的化療敏感度 …………………… 36
4-1. Eps8表現減少降子宮頸癌細胞之移行能力 …………… 38
4-2. Eps8對於子宮頸癌細胞FAK活化及表現的影響 ……… 38
4-3. Eps8增進子宮頸癌細中MMP-9之表現 …......................... 39
4-4. Eps8經由增加ERK活性而促進MMP-9之表現 ............. 39
4-5. ERK-MMP-9訊息路徑參與在Eps8媒介之細胞移行作用 … 40

第四章 討 論 …………………………………………….. 42
第五章 圖 表 …………………………………………… 45
參考文獻 …………………………………….................................. 76
著作 ................................................................................... 84
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