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研究生:陳姵妤
研究生(外文):Chen, Peiyu
論文名稱:研究在移型上皮細胞癌TSGH8301過度表現SOX2對細胞功能所造成的影響
論文名稱(外文):Investigating The Cellular Functions Of SOX2 In Urothelial Carcinoma Cell Line TSGH8301
指導教授:李沁李沁引用關係
指導教授(外文):Li, Chin
口試委員:黃憲斌李國榮
口試委員(外文):Huang, HsienBinLi, KuoJung
口試日期:2012-07-25
學位類別:碩士
校院名稱:國立中正大學
系所名稱:分子生物研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:64
中文關鍵詞:移型上皮細胞癌
外文關鍵詞:Urothelial carcinomaSOX2TSGH8301
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早期流行病學研究顯示移型上皮細胞癌在台灣西南沿海地區的盛行率高於其他地區,而這些地區同樣也是烏腳病盛行的區域,過去有文獻指出砷會導致烏腳病的發生,且暴露在低劑量的砷中也會提高罹患癌症的機率,因此早期認為飲用砷汙染的水源是導致烏腳病以及移型上皮細胞癌的主要原因。然而,檢閱先前的文獻卻發現罹患移型上皮癌的族群與罹患烏腳病的族群是不完全相同的,顯示出在台灣西南沿海地區除了砷汙染之外還有其他仍不清楚的分子機制也會導致移型上皮細胞癌的發生,近年來,許多研究亦指出異常的選擇性剪接為形成癌症的因素之一,因此,我們試圖去調查異常的選擇性剪接在移型上皮細胞癌中所扮演的角色。由實驗結果發現,來自台灣移型上皮細胞癌的細胞株TSGH8301以及BFTC905中有選擇性剪接異常的現象,同時,藉由生物晶片檢測也顯示出在移型上皮細胞癌細胞株TSGH8301以及BFTC905中有多種與RNA代謝相關的基因具有異常表達的情況,而SRY-related HMG-box 2 protein (SOX2) 則為其中之一。除此之外,我們也藉由in vivo CLIP assay發現SOX2為一種RNA結合蛋白質 (RNA binding protein)。根據研究指出雖然SOX2在許多癌症的發展過程中扮演了重要的角色,然而,SOX2真正導致致癌的分子機制至今仍是處於未知的階段。
為了探討SOX2作為一個RNA結合蛋白質對於移型上皮細胞癌造成的影響,首先,藉由lentiviral system在第二級的移型上皮細胞癌TSGH8301中建立具有SOX2過度表達的穩定細胞株,接著,利用西方墨點法的分析發現SOX2過度表現時,S100A14的表現量也隨之增加,推測會造成這樣的結果可能是因為SOX2會結合在S100A14 mRNA上進行後轉錄調節所導致的;此外,SOX2的過度表達也會經由縮短細胞週期時程而加快TSGH8301的生長速度,同時也減少了TSGH8301的移動能力。雖然SOX2的表現會加快細胞生長速度,但在實驗當中卻發現細胞週期各個階段的比例卻無顯著的不同。在未來的實驗中,我們將進一步探討SOX2的表現所造成TSGH8301生長速度上的差異現象原因為何,同時找出SOX2如何促進S100A14 mRNA的轉譯。

Early epidermiological studies had established that the counties of Southwestern Taiwan with unusually high prevalence of urothelial cell carcinoma (UC) are also those with high incidence frequency of Blackfoot disease. Nevertheless, close inspection of the earlier reports indicated that the populations with high incidence frequency of UC and of Blackfoot disease are different, implying that in addition to arsenic contamination other previously unidentified molecular mechanism is responsible for occurrence of UC in this region. Recent advances have indicated that alternative splicing could play a pivotal role in oncogenesis. In an effort to investigate the role of aberrant splicing in development of UC, we discovered that the urothelial cell carcinoma cell lines TSGH8301 and BFTC905 displays significant and clear alteration in their splicing activity. Through gene expression profiling, it was discovered that several proteins potentially functioning in RNA metabolism, including SRY-related HMG-box 2 protein (SOX2), is dysregulated in urothelial cell carcinoma cell lines. Moreover, we also identified the RNA binding activity of SOX2. Up-regulation of SOX2 is observed in brain, lung, breast, and bladder cancers and is generally correlated with poor prognosis. Although SOX2 plays an important role in initiation and progression of cancers, the molecular mechanism how SOX2 promotes oncogenesis is largely unclear. Hence, our goal is to investigate the role of SOX2 as RNA binding protein in oncogenesis of urothelial carcinoma.
The goal of my project is to identify the post-transcriptional mechanisms and the cellular events regulated by SOX2. The urothelial carcinoma cell line TSGH8301 exhibits little expression of SOX2 and is used as the model cell line. To investigate the cellular function of SOX2 in the the context of TSGH8301, I first established a SOX2-expressing TSGH8301 cell line using the lentiviral delivery system. At the molecular level, the expression of SOX2 greatly increases the protein level of S100A14, potentially through binding to the S100A14 mRNA. Furthermore, SOX2 appears to increase the growth rate by shortening cell cycle and also to decrease the migration ability of TSGH8301. However, although the growth rate of TSGH8301 is accelerated by the expression of SOX2, there is no significant difference in the percentages of the cells in each cell cycle stage. In the near future, we will confirm the phenomenon of SOX2-dependent increase to the growth of TSGH8301 and investigate how SOX2 promotes translation of S100A14 mRNA.

目錄
中文摘要 ……………………………………………………………………….I
英文摘要 ……………………………………………………………………..III
目錄 ………………………………………………………………………V
圖表&索引目錄 VIII
壹、 緒論 9
(一)、 移型上皮細胞癌 (Transitional cell carcinoma) 9
(二)、 後轉錄調節 (Post-transcriptional regulation) 10
(三)、 RNA結合蛋白質 (RNA binding protein ; RBP) 11
(四)、 SOX2 13
(五)、 S100A14 14
(六)、 研究動機與方向 14
貳、 實驗材料與方法 16
(一)、 材料 16
1. 質體來源 16
2. 抗體 16
3. 緩衝溶液配方 17
4. SDS-PAGE 配方 20
5. Tricine gel 配方 20
6. 細菌培養液 21
7. 細胞培養液 22
(二)、 方法 24
1. 質體構築 (plasmid construction) 24
2. 電穿孔質體轉型作用 (Transformation) 25
3. DNA萃取 (DNA extraction) 26
4. 細胞的轉染 (Transfection) 27
5. 慢病毒製備 (Production of VSVG-pseudotyped lentiviral particles) 28
6. 慢病毒感染 (Lentiviral infection) 28
7. 西方墨點法 (Western Blot) 29
8. 細胞免疫螢光染色法 (Immunofluorescence Staining) 29
9. RNA萃取 (RNA extraction) 30
10. 反轉錄聚合酶連鎖反應 (Reverse Transcription-PCR) 31
11. 即時定量聚合酶連鎖反應 (Real-time PCR) 31
12. 細胞生長速度測量 (Cell growth rate) 32
13. carboxyfluorescein diacetate, succinimidyl ester (CFSE)細胞增殖實驗 33
14. 利用Culture-Insert 進行細胞遷移能力分析 34
15. 細胞生長週期 (Cell cycle distribution) 34
參、 實驗結果 35
(一)、 建立具有Flag-SOX2過度表現的TSGH8301穩定細胞株 35
(二)、 建立具有SOX2過度表現的TSGH8301穩定細胞株 36
(三)、 在TSGH8301裡過度表達SOX2會影響S100A14表現量 37
(四)、 SOX2過度表現會影響TSGH8301的移動能力 38
(五)、 SOX2過度表現會影響TSGH8301的成長情形 38
(六)、 SOX2過度表現會影響TSGH8301的增殖速度 39
(七)、 SOX2過度表現不會影響TSGH8301的生長週期 40
肆、 討論 41
伍、 參考文獻 44
陸、 圖片說明 48
柒、 索引 60

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