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系統識別號 U0026-0709201013594800
論文名稱(中文) 探討 Oct4 在肺癌上調控雙特異性去磷酸酶 6 的角色
論文名稱(英文) Roles of Oct4 in Regulation of Dual-Specificity Phosphatase 6 (DUSP6) Expression and Its Relevance to Lung Cancer
校院名稱 成功大學
系所名稱(中) 生物化學暨分子生物學研究所
系所名稱(英) of Biochemistry and Molecular Biology
學年度 98
學期 2
出版年 99
研究生(中文) 吳姿錞
研究生(英文) Zih-Chun Wu
學號 s1697111
學位類別 碩士
語文別 中文
論文頁數 48頁
口試委員 指導教授-吳昭良
口試委員-賴明德
口試委員-蕭璦莉
口試委員-李哲欣
口試委員-張孟雅
中文關鍵字 肺癌  雙特異性去磷酸酶 6 
英文關鍵字 lung cancer  DUSP6 
學科別分類
中文摘要 Octamer 4 (Oct4) 是由 Pou5f1 基因轉錄的 POU 轉錄因子,在胚胎幹細胞中維持分化多能性 (plouripotency) 及自我更新 (self-renewal) 的能力。相對於不表現Oct4的體細胞,在不同種類腫瘤觀察到 Oct4 的表現,意味著 Oct4 在腫瘤形成過程中扮演重要角色。在膀胱癌小鼠模式中,Oct4 表現量的增加已證明會促進癌細胞的增生及轉移。雙特異性去磷酸酶6號 (dual-specificity phosphatase 6, DUSP6) 為絲裂原活化蛋白激酶 (mitogen-activated protein kinase, MAPK) 的去磷酸酶 (MAPK phosphatase, MKP) 之一,在胰臟癌中已知會造成細胞外訊息調節激酶2 (extracellular signal-regulated kinase 2, ERK-2) 的去活化,而被視為具有腫瘤抑制的功能。DUSP6 的表現與高復發且低存活率的非小細胞肺癌 (non-small cell lung cancer, NSCLC) 病患有密切的相關性。在未分化的小鼠胚胎幹細胞中利用染色質免疫沉澱分析,發現 DUSP6 可能是 Oct4 下游所調控的基因。在已發表的 cDNA 微陣列資料庫顯示,降低 Oct4 的表現會造成小鼠胚胎幹細胞中 DUSP6 的 mRNA 表現量降低。然而 Oct4 如何調控 DUSP6 表現的機制目前並不清楚。在本研究中,觀察肺癌病患的肺部組織切片染色及肺癌細胞株的結果,發現 Oct4 與 DUSP6 的表現量具有正相關性。在肺癌細胞株過度或降低 Oct4 的表現後,發現 DUSP6 在蛋白質及 mRNA 的表現量也隨之增減,可知 Oct4 與 DUSP6具有正向調控的關係。進一步確認 Oct4 如何在轉錄上調控 DUSP6 基因,我們利用染色質免疫沉澱分析及報導基因分析證明 Oct4是直接結合至 DUSP6 啟動子而增加其活性,以調控DUSP6 基因的表現。在細胞實驗結果也觀察到 Oct4 會增加肺癌細胞的生長速度及轉移能力,並在不同 Oct4 表現量的實驗組中發現 DUSP6 在肺部組織表現的位置與 Oct4 表現的位置相似。綜合以上結果推測 Oct4 促進非小細胞肺癌轉移的機制,可能是藉由調控 DUSP6 基因的表現,啟動一連串的訊息傳遞而影響非小細胞肺癌的轉移。目前 DUSP6 在轉移所扮演的角色尚未明確,需進一步在細胞及動物實驗中將 Oct4 過量表現並降低 DUSP6 的表現量下,觀察肺癌細胞的爬行及侵入能力是否會受到影響,以證實 Oct4 確實可透過調控 DUSP6 而影響肺癌細胞的轉移。
英文摘要 Octamer 4 (Oct4) is a POU domain-containing transcription factor encoded by Pou5f1 and required to maintain the pluripotency and self-renewal of embryonic stem (ES) cells. However, Oct4 has been detected in several human tumors, suggesting a potentially critical role in tumorigenesis. Dual-specificity phosphatase 6 (DUSP6) is one of the mitogen-activated protein kinase (MAPK) phosphatases (MKPs) and its inactivation of extracellular signal-regulated kinases 2 (ERK-2) is believed as to function as a tumor suppressor in pancreatic cancer. In contrast, DUSP6 is closely associated with an increased risk of recurrence and decreased overall survival among patients with non-small cell lung cancer. Moreover, chromatin immunoprecipitation (ChIP) assay revealed that DUSP6 may be a potential downstream target of Oct4 in undifferentiated mouse ES cells. Down-regulation of DUSP6 mRNA expression in Oct4-knockdown mouse ES cells was also demonstrated in the public domain database. However, the mechanism underlying the regulation of DUSP6 expression by Oct4 is still unclear. The aim of this study was to study the association of Oct4 and DUSP6 in lung cancer and to elucidate its mechanism of action. My results revealed that there was a positive correlation between the expression of Oct4 and DUSP6 in lung cancer cells. Overexpression of Oct4 enhanced the expression of DUSP6 at both mRNA and protein levels. Chromatin immunoprecipitation (ChIP) and reporter assays showed that Oct4 enhanced the promoter activity of DUSP6 through direct binding to its promoter to regulate DUSP6 gene expression. Oct4 also enhanced the proliferation rate and migratory ability of lung cancer cells in vivo. Taken together, these results suggest that Oct4 regulates DUSP6 expression to initiate signal transduction cascades, leading to promoting the metastasis of non-small lung cancer cells. In the future, I will focus on studying the effects of Oct4-mediated DUSP6 expression on enhancing tumor migration and invasion in vitro and in vivo.
論文目次 考試合格證明 I
中文摘要 II
英文摘要 IV
誌謝 VI
圖目錄 X
縮寫 XI
一、緒論 1
1.肺癌 (Lung Cancer, Bronchogenic carcinoma) 1
2. Octamer 4 (Oct4) 2
2.1. Oct4 的結構及功能 2
2.2. Oct4 與腫瘤 3
2.3. Oct4 的調控基因 5
3. 雙特異性去磷酸酶 (Dual-Specificity Phosphatase 6, DUSP6) 6
3.1. DUSP6的結構及功能 6
3.2. DUSP6 與腫瘤 6
二、研究動機 8
三、材料與方法 9
1. 材料 9
1.1. 臨床檢體 9
1.2. 質體 9
1.2.1. 質體 9
1.2.1. RNA干擾質體 (short hairpin RNA, shRNA) 10
1.3.細胞株 11
1.4.抗體 12
1.4.1. 一級抗體 12
1.4.2. 二級抗體 12
1.5.引子 13
1.6.實驗動物 13
2. 方法 14
2.1. 細胞株 14
2.2. 西方墨點法 (Western blot analysis) 14
2.3. 免疫組織化學染色 (Immunohistochemistry assay, IHC) 15
2.4. 質體轉染 ( Transcient transfection) 15
2.5. 慢病毒生產及感染 (Lenti-virus production and infection) 16
2.6. 反轉錄酶聚合連鎖反應 (Reverse transcriptase polymerase chain reaction, RT-PCR ) 17
2.7. 即時半定量反轉錄酶聚合連鎖反應 (Real time RT-PCR) 18
2.8. Luciferase reporter assay (啟動子活性分析) 18
2.9. Chromatin Immunoprecipitation, ChIP (染色質免疫沉澱法) 19
2.10. 動物實驗 21
2.11. Boyden chamber assay 21
2.11. 統計分析 22
四、結果 23
1. 在肺癌病患的肺部組織切片及非小細胞肺癌細胞株中,Oct4 與 DUSP6 表現量具有正相關性。 23
2. Oct4 及 DUSP6 存在正向調控關係。 23
3. Oct4 直接結合至 DUSP6 啟動子,並增加 DUSP6 啟動子活性。 24
4. Oct4促進腫瘤生長速度並增加其轉移能力。 25
5. 動物實驗證明 Oct4 及 DUSP6 表現具有相互調控關係。 25
6. Oct4 藉由 DUSP6 增加腫瘤細胞的爬行能力。 26
五、討論 27
參考文獻 31
圖 37
附錄一、本實驗室建構之質體 46

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