CTEN 是 tensin 家族的成員之一，位於 focal adhesion ，主要參與細胞貼附、增生及遷移等生理功能。正常細胞中的 CTEN 表現量較低且主要位於細胞質及focal adhesion，但在大腸直腸癌與發炎大腸組織的細胞中，CTEN 不只表現量上升，大量 CTEN 也被發現聚集在細胞核內，然而細胞核中 CTEN 的功能尚未完全了解，我們推測癌細胞核中的 CTEN 可能與腫瘤形成有關。為了檢驗 CTEN 在細胞中不同的分布是否影響腫瘤形成的特性，我們將 CTEN 加上特定的定位胺基酸序列，包含 Src myristoylation signal (Myr) 以及 nuclear localization signal (NLS)，再轉染至與大腸癌細胞株 HCT116 中表現。由實驗結果發現表現 NLS-CTEN 的 HCT116 細胞比表現 WT-CTEN 的細胞，有明顯較多的 CTEN 在細胞核內，其細胞遷移與侵襲的能力皆顯著提升。同時，我們也比較表現 WT-CTEN 與 NLS-CTEN 的HCT116 細胞轉錄體的差異，以探尋受細胞核CTEN 影響差異性表現的下游基因，以瞭解其影響腫瘤形成特性的分子機制。另一方面，由於轉錄因子 NF-κB 是連接腸道發炎與大腸癌發生的關鍵因子，因此我們研究細胞核 CTEN 是否參與 NF-κB 訊息路徑。我們先前的研究發現，經由 TNF 刺激後，細胞核中 CTEN 和 NF-κB 次單元體 p65 交互作用明顯增加，在大腸癌細胞株 knockdown CTEN 後，會使 NF-κB 轉錄活性下降以及減少一些下游基因的表現。本論文利用 GST pull-down assay 尋找 CTEN 蛋白質上與 p65 結合的重要區塊，並檢驗 CTEN 是否會影響 p65 結合 DNA 的活性，以研究 CTEN 調節 NF-κB 訊息路徑的分子機制。

　C-terminal tensin-like (CTEN) belongs to tensin family that localizes to focal adhesion. It involves in cell adhesion, proliferation and migration. Previous studies have demonstrated that CTEN is highly expressed and accumulated in the nucleus of colon cancer cells and inflamed colonic tissues whereas it mainly localizes in cytoplasm and focal adhesion in normal cells. The presence of CTEN in the nucleus, which is not observed in normal tissues, suggests that nuclear CTEN may have oncogenic function and is associated with tumorigenesis in colon cancer. To identify the relationship between subcellular localization and oncogenic properties of CTEN, CTEN variants with specific localization motifs, including the Src myristoylation signal (Myr) and the nuclear localization signal (NLS), were transfected to colon cancer cell HCT116. We have found that the ability of cell migration and invasion were promoted in NLS-CTEN-expressed HCT116 cells comparing to WT-CTEN expressed ones. Meanwhile, we also analyzed the transcriptome of HCT116 cells expressing WT-CTEN and NLS-CTEN to identify the differential expressed downstream genes affected by nuclear CTEN and to understand the molecular mechanism of nuclear CTEN in cell tumorigenicity. On the other hand, transcription factor NF-κB is a critical link between inflammation and colon cancer, we therefore investigate whether nuclear CTEN participates in NF-κB signaling. Our previous studies have also demonstrated that CTEN interacts with NF-κB subunit p65 in the nucleus after TNF stimulation. Knockdown of CTEN down-regulates the transcriptional activity of NF-κB and the expression of certain downstream genes. In this thesis, glutathione S-transferase (GST) pull-down assay was used to map the p65 binding region on CTEN and whether CTEN affects the DNA binding activity of p65 was also examined to study the molecular mechanism of CTEN in NF-κB signaling regulation.

目錄. I
縮寫表 IV
摘要 . VI
Abstract VII
一、 研究背景 1
1.1 Focal adhesion 1
1.2 Tensin family 1
1.3 C-terminal tensin-like protein (CTEN) 之功能研究 2
1.4 CTEN 穿梭於細胞核與細胞質間之分子機制 3
1.5 Nuclear Factor kappa B (NF-B) 訊息傳導路徑 4
1.6 本論文之研究目的 5
二、 材料與方法 7
2.1 菌株 7
2.2 質體DNA 7
2.3 DNA分析與質體建構 8
2.3.1 聚合酶連鎖反應 (polymerase chain reaction, PCR) 8
2.3.2 DNA 片段純化 9
2.3.3 限制酶切質體與PCR產物 9
2.3.4 DNA 接合 (DNA ligation) 9
2.3.5 重組質體轉型與篩選 9
2.3.6 質體純化 10
2.3.7 DNA瓊脂糖膠體電泳 10
2.4 RNA 分析 10
2.4.1 RNA 純化 10
2.4.2 次世代定序 (next generation sequencing, NGS) 及差異表現基因分析 10
2.4.3 製備cDNA 11
2.4.4 定量 PCR (quantitative PCR, qPCR) 11
2.5 細胞相關實驗 11
2.5.1 細胞培養 11
2.5.2 細胞繼代 12
2.5.3 細胞計數 13
2.5.4 細胞冷凍保存 13
2.5.5 細胞解凍 13
2.5.6 細胞轉染 (Transfection) 13
2.5.7 si-RNA knockdown 基因表現 14
2.5.8 TNF 處理 14
2.5.9 細胞增生分析 Proliferation assay 14
2.5.10 細胞遷移分析 Migration assay 15
2.5.11 細胞侵襲分析 Invasion assay 15
2.6 全細胞蛋白質萃取 16
2.7 細胞核、細胞質蛋白質之分離 16
2.8 GST重組蛋白質表現 17
2.9 GST pull down assay 17
2.10 p65 DNA binding activity 17
2.11 蛋白質分析 18
2.11.1 蛋白質膠體電泳 18
2.11.2 西方墨點法 18
三、 研究結果 20
3.1 改變外源 CTEN 蛋白質在 HCT116 細胞中的分布 20
3.2 分析 CTEN 在癌細胞中分布的差異對腫瘤形成特性的影響 21
3.3 外源表現 WT-CTEN 與 NLS-CTEN 細胞的轉錄體分析 21
3.3.1 以 NGS 分析轉錄體 (transcriptome) 21
3.3.2 以 qPCR 檢測差異表現基因 22
3.4 分析 CTEN 蛋白質上與轉錄因子 p65 結合的區域 23
3.4.1 以大腸桿菌表現帶有 GST tag 之短片段 CTEN 23
3.4.2 進行 pull-down assay 找出 CTEN 上結合 p65 的胺基酸片段 24
3.4.3 以大腸桿菌表現帶有 GST tag 之較長的 CTEN 區域 25
3.4.4 進行 pull-down assay 找出 CTEN 上結合 p65 的區域 25
3.5 分析 CTEN 是否影響 p65 結合目標 DNA 的活性 26
四、 討論與未來方向 28
4.1 細胞核中 CTEN 影響腫瘤形成特性之機制 28
4.2 細胞核中 CTEN 調控 NF-κB 訊息路徑之分子機制 30
五、 參考文獻 33
六、 圖與表 40

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