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研究生:徐漢民
研究生(外文):Han-Min Hsu
論文名稱:探討轉譯後修飾作用對STAT3活化之影響
論文名稱(外文):STAT3 activation mediated through posttranslational modifications
指導教授:張久瑗
指導教授(外文):Jeou-Yuan Chen
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生命科學暨基因體科學研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:78
中文關鍵詞:癌症細胞增生訊息傳遞與活化因子
外文關鍵詞:STAT3CD44
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STAT3的是一個潛在的轉錄因子,其下游目標基因之功能與血管新生 、胞週期進行、細胞遷移及抑制免疫反應有關,因此在癌症細胞中,經常發現STAT3持續且過度地活化。傳統的STAT3活化路徑是經由細胞因子或生長因子的刺激,促進STAT3 Y705的磷酸化和同源二聚體之形成後,進入細胞核中啟動下游基因的表現。據文獻報導,STAT3二聚體的形成及轉錄功能也受到K685乙醯化的調控。在我們以前的研究中,我們以造骨蛋白(Osteopontin, OPN)引起CD44(第一型穿膜蛋白,促進細胞遷移和存活)聚合時,藉由細胞內吞作用,CD44會進入細胞核中與STAT3相互作用,此條件下之STAT3具有乙醯化而非磷酸化修飾,與一般文獻報導的磷酸化促進STAT3進核不同,因此在本論文中,我探討STAT3磷酸化及乙醯化扮演的角色和基因調控上的差異。我比較IL-6處理下與CD44聚合所引起的磷酸化及乙醯化,在不同時間測量內生性p-STAT3和ac-STAT3的變化,發現IL-6可促進磷酸化及乙醯化發生,而CD44聚合只促進乙醯化發生,在此二種處理下,皆發現STAT3進核增加及細胞增生的現象。另外我發現磷酸化與乙醯化可以獨立發生,並同時存在於相同STAT3蛋白;此外,我以IL-6刺激STAT3野生型(WT)、乙醯化突變型(K658R),磷酸化突變型(Y705F)或雙點突變型(K685R/Y705F)後,進行報導基因分析,發現磷酸化突變時,STAT3活性降低,乙醯化突變時STAT3活性不變,證實p-STAT3與ac-STAT3可結合在不同DNA序列。進一步由染色質免疫沉澱分析得知p-STAT3與ac-STAT3在cyclin D1的啟動子具有不同的偏好結合位。根據這些結果,我們認為 CD44聚合與IL-6引起STAT3活化的路徑不同,為了證明STAT3可藉由磷酸化或乙醯化調節不同的基因,我利用反轉錄病毒建立了穩定表現STAT3野生型和STAT3突變型之小鼠胚胎纖維母細胞株,利用染色質免疫沉澱後定序之技術,試圖偵測由p-STAT3或ac-STAT3調控的基因。
The members of signal transducer and activator of transcription (STAT) family are a group of latent transcription factors. In response to cytokines and growth factors, they are post-translationally modified which allows them to form dimmers and translocate to the nucleus to regulate gene expression. Stat3 is a multifaceted oncogene, which is frequently activated in many human cancers. Stat3 regulates the expression of a variety of genes in response to cell stimuli, and plays a key role in a plethora of cellular processes such as cell growth and apoptosis. It is well established that the dimerization of Stat3 is mediated through the phosphorylation of Y705. Acetylation of K685 has also been reported to promote Stat3 dimerization. In our previous study, we have demonstrated that ligation of CD44, a type I transmembrane protein known to be involved in cell/cell and cell/matrix interactions, promotes acetylation of Stat3. The acetylated Stat3 forms homodimer and translocates into the nucleus, where it promotes cell proliferation through transactivation of cyclin D1. It is noted that CD44-mediated Stat3 activation is independent of cytokine- as well as growth factor-mediated phosphorylation process. To gain further understanding of the molecular mechanisms involving Stat3 activation, in this study, I further characterize the activation and transcriptional activity of phosphorylated and acetylated Stat3. In HeLa cells, I showed that IL-6 treatment leads to phosphorylation and acetylation of the endogenous Stat3, whereas ligation of CD44 by anti-CD44 monoclonal antibody Hermes-3 induces Stat3 acetylation. Both IL-6- and CD44 ligation-induced Stat3 modification promotes nuclear localization of Stat3. In addition, I have overexpressed the wild-type, phosphorylation-deficient (K685R), acetylation-deficient (Y705F), and double mutants in the Stat3-null prostate cancer PC-3 cells and the Stat3-knockout mouse embryonic fibroblasts (MEF). Similarly, the ectopically expressed Stat3(WT) can be phosphorylated and acetylated under IL-6 treatment, while Stat3(Y705F) is acetylated, Stat3(K685R) is phosphorylated, and Stat3(Y705F/K685R) is not modified. To further examine the transcriptional activity, vector encoding the individual Stat3 was co-transfected into PC-3 cells with an sis-inducible element (SIE)-directly luciferase reporter plasmid. My data showed that Stat3(WT) and Stat3(K685R) can transactivate the expression of luciferase to the same extent in response to Il-6 treatment, but Stat3(Y705F) failed to do so. Furthermore, chromatin immunoprecipitation (ChIP) assay revealed that Stat3(Y705F) and Stat3(K685R) have distinct binding preference towards the Stat consensus sequences on cyclin D1 promoter. These data clearly demonstrate that phosphorylated and acetylated Stat3, although both are capable of regulating gene expression, display varying DNA binding activity and transcriptional activity. This study thus paths the groundwork for further characterization of the function of Stat3 upon differential modification.
目錄
項目 頁碼
目錄 I
圖目錄   V

縮寫表 VI

中文摘要 VII

英文摘要 VIII

緒論
一、 預防細胞癌化的機制 1
1. 細胞週期的調控 1
2. 細胞凋亡 2
二、 癌細胞的特性(Hallmarks of cancer) 3
1. 自足的生長訊息 4
2. 對抑制生長訊息不敏感 4
3. 抵抗細胞凋亡 4
4. 血管新生 5
5. 細胞侵犯與轉移 6
6. 無限制的細胞分裂能力 7
三、 Signal transducer and activator of transcription family 7
1. 細胞激素訊息傳導 8
2. STAT 8
3. STAT3的功能 9
4. STAT3的磷酸化修飾 10
5. STAT3的乙醯化修飾 10
6. 非組蛋白的蛋白質乙醯化 11
四、CD44(cluster of differentiation 44) 12
1. CD44的簡介 12
2. CD44的功能 14
3. 癌症幹細胞(caner stem cell) 14

研究目標 16

材料與方法
一、 癌症細胞與培養 18
二、 PCR 18
三、 細胞轉染反應 19
四、 SDS-PAGE 19
五、 西方墨點法 20
六、 免疫沉澱 21
七、 報導基因分析法 21
八、 染色質免疫沉澱分析 22
九、 免疫螢光染色 24
十、 使用STAT3shRNA抑制(Knockdown)STAT3表現 24

實驗結果
一、 探討CD44聚合及IL-6所引發的STAT3修飾作用 26
1. IL-6同時促進STAT3的磷酸化及乙醯化 26
2. IL-6促進p-STAT3及ac-STAT3轉移並累積在細胞核中 27
3. CD44聚合促進STAT3的乙醯化 27
4. CD44聚合促進ac-STAT4在細胞核中的累積 28
5. 外生性的STAT3磷酸化及乙醯化 29
6. 建立穩定表現野生型及不同突變型STAT3的老鼠胚胎纖維母細胞株
30
二、CD44聚合及IL-6對細胞的影響 30
1. IL-6及CD44聚合能促進細胞生長 30
2. Hermes-3引發的CD44聚合能促進細胞生長 31
3. IL-6及CD44聚合經由活化STAT3促進細胞生長 32
4. p-STAT3與ac-STAT3促進細胞生長 33
三、探討p-STAT3及ac-STAT3調控基因的能力 34
1. ac-STAT3及p-STAT3調控基因轉錄的差異 34
2. CD44聚合及IL-6促進STAT3的修飾並有不同的偏好結合序列 35

討論
一、 探討IL-6或是CD44所促進的修飾作用的差異 36
二、 探討IL-6或是CD44聚合所促進的STAT3進核 37
三、 探討STAT3磷酸化及乙醯化發生的獨立性 38
四、 探討p-STAT3及ac-STAT3結合序列的差異 38
五、 探討p-STAT3及ac-STAT3的生理意義 39

參考文獻 41

附圖 48

附錄
附圖一、CD44聚合後進入細胞核並促進STAT3的乙醯化 62


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