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研究生:翁而瑩
研究生(外文):Erh-Ying Weng
論文名稱:研究 SOX2 依賴的 S100A14 其表現對細胞功能所造成的影響
論文名稱(外文):Investigating the cellular functions of SOX2-dependent S100A14 expression
指導教授:李沁李沁引用關係
指導教授(外文):Chin Li
口試委員:黃憲斌李國榮
口試委員(外文):Hsien-Bin HuangKuo-Jung Li
口試日期:2013-07-18
學位類別:碩士
校院名稱:國立中正大學
系所名稱:生物醫學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:55
中文關鍵詞:S100A14SOX2
外文關鍵詞:S100A14SOX2
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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,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。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。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) 並且會結合在S100A14的mRNA上;並且利用西方墨點法發現當抑制SOX2 表現時會去調結S100A14在轉錄後調節機制的轉譯功能。
為了探討S100A14作為一個被SOX2 RNA結合蛋白質所結合的RNA對於移型上皮細胞癌造成的影響。首先,藉由lentiviral system在第三級的移型上皮細胞癌BFTC905中建立具S100A14表達受到抑制的穩定細胞株,接著,利用西方墨點法的分析發現S100A14表現受到抑制時,SOX2的表現量也隨之抑制;此外,抑制S100A14表達也會增加了BFTC905的移動能力;雖然抑制S100A14的移動能力表現會加快,但在實驗當中卻發現細胞生長速度及細胞週期各個階段的比例卻無顯著的不同。另一方面,為了進一步去確認SOX2在轉錄後修飾中調控基因中所扮演的功能,利用了細胞融合試驗 (Heterokaryon assay) 去証實SOX2為一個穿梭蛋白。在目前的實驗結果中,我們發現抑制SOX2會影響S100A14的蛋白質表現量,而在未來實驗中,則要得知是否過度表現SOX2會影響S100A14的蛋白質表達,因而引響BFTC905移動能力;進而去証實SOX2使癌細胞惡化不是透過細胞增生而是由於細胞的侵襲和轉移。除此之外,還製備 SOX2 抗體以供之後研究需要。
Early epidermiological studies had established that the counties of Southwestern Taiwan exhibit unusually high prevalence of urothelial cell carcinoma (UC). In an effort to investigate the role of aberrant splicing in development of UC, the study conducted in our laboratory demonstrated 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 up-regulated in urothelial cell carcinoma cell lines. The research carried out in our laboratory has identified that SOX2 likely plays dual functions both as transcription factor and as RNA binding protein. In addition, we discovered that SOX2 binds to the S100A14 mRNA in vivo and that knock-down of SOX2 expression leads modulates S100A14 translation through a post-translational regulatory mechanism. Hence, my primary goal is to investigate the cellular function of S100A14. In order to achieve my goal, I first established a S100A14 knocked-down BFTC905 cell line using the lentiviral delivery system. At the molecular level, the anti-S100A14 short hairpin RNA (shRNA) effectively decreases both the mRNA and the protein level of S100A14. Furthermore, suppression of S100A14 expression appears to increase the migration ability of BFTC905. Although the overall growth rate of BFTC905 appears to remain similar upon the expression of anti-S100A14 shRNA, the percentage of the cells in the S stage increases significantly, suggesting an alteration to the cell cycle progression. On the other hand, to provide additional additional evidences that SOX2 functions at the post-transcriptional gene regulation, I performed the heterokaryon assay to determine SOX2 is in fact a shuttling protein. With the findings that knock-down of SOX2 leads to an increase to the protein output of the S100A14 mRNA, we proposed that expression of SOX2 in BFTC905 modulates S100A14 protein expression, an event in turn increasing cell mobility. Hence, expression of SOX2 likely promotes cancer malignancy not through increasing cell growth but through increasing tumor invasiveness and metastasis. Additional to these works, I will also purify recombinant SOX2 protein for generation of the anti-SOX2 antibody, which will be essential for future study.
中文摘要 I
英文摘要 III
目錄 V
圖表目錄 VIII
壹、 緒論 1
一、 移型上皮細胞癌 (Transitional cell carcinoma) 1
二、 後轉錄調節 (Post-transcriptional regulation) 2
三、 RNA結合蛋白質 (RNA binding protein ; RBP) 3
四、 SOX2 4
五、 S100A14 5
六、 研究動機與方向 6
貳、 實驗材料與方法 7
一、 材料 7
1. 質體來源 7
2. 抗體 8
3. 緩衝溶液配方 8
4. SDS-PAGE 配方 12
5. 細菌培養液 12
6. 細胞培養液 13
二、 方法 16
1. 質體構築 (plasmid construction) 16
2. 電穿孔質體轉型作用 (Transformation) 17
3. DNA萃取 (DNA extraction) 18
4. 細胞的轉染 (Transfection) 20
8. 慢病毒製備 (Production of VSVG-pseudotyped lentiviral particles) 23
9. 慢病毒感染 (Lentiviral infection) 23
10. 西方墨點法 (Western Blot) 24
11. 細胞免疫螢光染色法 (Immunofluorescence Staining) 24
12. RNA萃取 (RNA extraction) 25
13. 反轉錄聚合酶連鎖反應 (Reverse Transcription-PCR) 26
14. 即時定量聚合酶連鎖反應 (Real-time PCR) 26
15. 細胞生長速度測量 (Cell growth rate) 27
16. carboxyfluorescein diacetate, succinimidyl ester (CFSE)細胞增殖實驗 28
17. 利用culture-insert 進行細胞遷移能力分析 29
18. 細胞生長週期 (Cell cycle distribution) 29
參、 實驗結果 30
一、 建構pET15b-deltaHMGSOX2 30
二、 利用細胞融合試驗證明SOX2有出核現象 30
三、 建立抑制S100A14表現的BFTC905穩定細胞株 31
四、 抑制S100A14會影響SOX2表現量在BFTC905中 32
五、 抑制S100A14表現會影響BFTC905的移動能力 32
六、 BFTC905的成長速度不會因抑制S100A14表現所影響 33
七、 抑制S100A14表現不會影響BFTC905的增殖速度 33
八、 抑制S100A14表現影響BFTC905的生長週期 34
肆、 討論 35
伍、 參考文獻 38
陸、 圖片說明 41

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