我們的研究探討了STMN3的功能和調節機制，STMN3是膀胱癌中的微管去穩定磷酸蛋白。我們的結果顯示穩定轉染pSTMN3-HaloTag在J82會上調STMN3 mRNA和STMN3蛋白質表現，而在BFTC905中使STMN3基因沉默會下調STMN3 mRNA和STMN3蛋白質表現。過度表達STMN3會誘導細胞增殖並改變細胞週期調節相關的蛋白質表現，而基因沉默則會抑制細胞增殖並改變細胞週期調節相關的蛋白質表現。STMN3蛋白上的絲氨酸50、65及73位置的突變會降低J82細胞中的細胞增殖和細胞非貼附性生長。在STMN3中加入LY294002 (PI3K抑制劑)和AZD6244 (MAP2K抑制劑)會穩定乙酰化微管蛋白的表現。對STMN3蛋白中幾種潛在的磷酸化位點的突變會略微降低J82細胞中的肌動蛋白聚合。我們得出結論，STMN3在膀胱癌中扮演致癌基因的角色，並且可能受PI3K與MEK/ERK訊息傳遞路徑所調控，也能干擾微管及肌動蛋白穩定性。

Our study explored the function and regulatory mechanisms of STMN3 which is the microtubule-destabilizing phosphoprotein in the bladder cancer. Our results showed that stable transfection of the pSTMN3-HaloTag plasmid upregulates while knockdown of the STMN3 gene suppresses STMN3 mRNA and STMN3 protein levels in J82 and BFTC905 cells, respectively. Exogenous STMN3 expression induces, while knockdown suppresses cell proliferation and alters the protein expression levels of several cell cycle regulators. Mutations on serine 50, 65 or 73 of the STMN3 protein decreases cell proliferation and colony formation/anchorage-independent cell growth in J82 cells. Inhibition of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and mitogen-activated protein kinase kinase (MAP2K) stabilize acetylated-tubulin. Mutagenesis on several potential phosphorylated serines in the STMN3 protein slightly decrease actin polymerization in J82 cells. Taken together, our data indicated that STMN3 functions as an oncogene in bladder cancer, and regulated by PI3K and MEK/ERK signaling pathways, also interfered microtubule and actin polymerization stability.

論文審定書 i
致謝 ii
摘要 iii
Abstract iv
英文縮寫表 v
目錄 vi
圖次 ix
表次 x
壹、 緒論 (Introduction) 1
一、 膀胱癌 (Urinary bladder urothelial carcinoma, UBUC) 1
二、 微管 (Microtubule, MT) 3
三、 Stathmin 3 (STMN3) 4
貳、 實驗材料與方法 (Materials and methods) 6
一、 細胞培養 6
(一) 膀胱癌細胞株 (Cells and cell culture) 6
(二) 細胞解凍 (Thawing frozen cells) 6
(三) 繼代培養 (Subculture adherent cells) 7
(四) 細胞冷凍保存 (Freezing cells) 7
二、 質體製備 8
(一) 質體 (Plasmid) 8
(二) 瓊脂膠體電泳 (Agarose gel electrophoresis) 8
(三) 膠體DNA純化 (Gel extraction) 9
(四) 質體轉型 (Transformation) 9
(五) 質體純化 (Plasmids purification) 9
三、 基因轉染 11
(一) 瞬時轉染 (Transient transfection) 11
(二) 慢病毒感染 (Lentiviral infection) 11
四、 cDNA製備與Quantitative real-time (qPCR) 12
(一) Total RNA萃取 (Total RNA extraction) 12
(二) 反轉錄聚合酶連鎖反應 (Reverse transcription) 13
(三) 定量即時聚合酶鏈鎖反應 (Quantitative real-time; qRT-PCR) 13
五、 蛋白質電泳與西方墨點法 13
(一) 蛋白質純化 (Protein purification) 13
(二) 蛋白質定量 (Bradford protein assay) 14
(三) 蛋白質電泳 (SDS-PAGE) 14
(四) 西方墨點法 (Western Blot) 15
六、 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay 15
七、 5-bromo-2-deoxyuridine (BrdU) assay 16
八、 Cell proliferation (Fluorometric) 16
九、 細胞週期 (Cell cycle analysis) 17
十、 細胞遷移及侵犯試驗 (Transwell migration and invasion assay) 17
(一) 細胞遷移試驗 (Migration assay) 17
(二) 細胞侵犯試驗 (Invasion assay) 18
十一、 細胞轉型分析 (Soft agar assay) 18
十二、 定點突變 (Site-directed mutagenesis) 19
十三、 Actin Polymerization/Depolymerization (Fluorometric) 20
(一) Actin polymerization assay 20
(二) Actin depolymerization assay 21
十四、 實驗數據統計分析 21
參、 結果 (Results) 28
一、 分析人類膀胱癌細胞株之內生性STMN3的mRNA與蛋白質表現 28
二、 高度表現STMN3會促進膀胱癌細胞增生能力，抑制STMN3表現會抑制膀胱癌細胞增生能力 28
三、 高度表現STMN3會促進細胞週期進展，抑制STMN3表現會使細胞週期停滯在G2/M期 29
四、 高度表現STMN3會促進膀胱癌細胞非貼附性生長能力，抑制STMN3表現會抑制膀胱癌細胞非貼附性生長能力 30
五、 高度表現STMN3會促進膀胱癌細胞遷移及侵犯能力抑制STMN3的表現會抑制膀胱癌細胞遷移及侵犯能力 31
六、 STMN3磷酸化位點S50, S65, S73的突變會抑制膀胱癌細胞增生能力及細胞非貼附性生長能力 31
七、 STMN3會受到PI3K及MAPK路徑調控並穩定微管蛋白 (tubulin)的表現，且STMN3磷酸化位點S50, S65, S73的突變會影響微管蛋白的穩定 33
八、 STMN3磷酸化位點S50, S65, S73的突變會抑制膀胱癌細胞肌動蛋白 (actin)的聚合 34
肆、 討論 (Discussion) 50
伍、 參考文獻 (Reference) 52
陸、 Supplementary Data 56

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