摘要
TCF12蛋白是第一類螺旋-環-螺旋 (helix-loop-helix) 蛋白質家族的一個成員，會直接結合到下游基因的啟動子上之E-box序列來調控基因之表現。TCF12蛋白在28%大腸直腸癌患者之腫瘤組織中有過量表現的情形，並與這些病人發生轉移有關，最近我們實驗室進一步發現TCF12蛋白促使大腸癌細胞惡化可能是因其能誘發腫瘤細胞的幹細胞特性 (cell stemness)。氯硝柳胺 (niclosamide) 是一種使用超過50年的抗絛蟲藥，幾年前被篩選出具有抑制癌幹細胞的能力，可以抑制相關的信號途徑如Wntβ-catenin，但是否能抑制TCF12蛋白所誘導的癌幹細胞特性則還是未知的，因此我採用了實驗室所建立的過度或降低TCF12表現的大腸直腸癌細胞株來分析其對氯硝柳胺的感受性，結果發現TCF12表現量高的癌細胞對於傳統化療藥物5-FU有較高的抵抗力，但對氯硝柳胺則較為敏感；而對於降低TCF12表現的細胞則呈現相反的結果。TCF12過度表現的癌細胞中，CD133、CD326、CD44、ALDH1、ABCG2等與癌幹細胞特性有關的基因 mRNA表現也是明顯增加，CD133、CD326、CD44在細胞表面上的量，以及這些細胞在無血清及無附著條件下形成球狀體的能力亦都明顯提高，但當處理以氯硝柳胺，這些TCF12所誘導的癌幹細胞特性都被大幅地抑制。實驗室先前的研究已發現MALAT1會與TCF12結合，Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) 是一種長鏈非編碼RNA (long non-coding RNA, lnc RNA)，在很多癌症中都會過量表現並且會導致腫瘤轉移與惡化。在我的RNA免疫共沉澱實驗中，氯硝柳胺的處理下卻能有效抑制TCF12與MALAT1的結合。另外，我的E-box pulldown實驗中，也發現在MALAT1啟動子的E-box上TCF12與另一轉錄因子Oct-4的互動可受到氯硝柳胺處理的干擾，這可以說明氯硝柳胺處理 24小時TCF12量沒有明顯改變但MALAT1卻表現量降低的現象。另外，也有研究指出MALAT1會藉由Wnt/β-catenin信號途徑誘導癌幹細胞特性。我發現在TCF12高表現的大腸癌細胞中，除了MALAT1的表現量較高，其細胞核內β-catenin及下游基因cyclin D1及c-Myc也都有較高表現，這些現象也能夠在投藥氯硝柳胺後被抑制。綜合結果指出TCF12的過量表現會明顯提高大腸直腸癌的癌幹細胞特性，並且TCF12可能藉由調控MALAT1使Wnt/β-catenin信號途徑也大量表現來誘導癌幹細胞特性，這些由TCF12誘導的作用則能夠被氯硝柳胺所抑制，這不僅闡明了部分TCF12MALAT1促進腫瘤惡化的機制，同時也提供了一個有潛力的治療目標。

關鍵字: TCF12、MALAT1、氯硝柳胺、Wnt/β-catenin信號途徑、癌幹細胞特性

Abstract
TCF12 is a class I member of helix-loop-helix protein family, regulates downstream genes via directly binding to E-box motifs, and contributes to cancer cell epithelial-to-mesenchymal transition and gain-of-stemness. Niclosamide is a reported inhibitor of cancer stem cells, which can suppress many cancer stemness-related signal cascades including the Wnt/β-catenin pathway. However, it remains to be defined whether niclosamide also has the activity to repress TCF12-mediated stemness in colorectal cancer (CRC). Using our established CRC cell clone with ectopic TCF12 expression, we observed that TCF12 overexpression increased CRC cell susceptibility to niclosamide even though it conferred ceullular resistance to the conventional chemotherapeutic agent 5-fluorouracil. Reciprocally, knockdown of TCF12 expression would reduce CRC cell sensitivity to niclosamide but elevate cell resistance to 5-fluorouracil. On the other hand, expressions of TCF12 and stemness markers CD133, CD326, CD44, ALDH1, and ABCG2 were significantly repressed in niclosamide-treated CRC cell lines by a concentration- and time-dependent manner. The cell-surface levels of CD133, CD326, and CD44 and the cell-spheroid formation levels were also consistently reduced. These results together suggested that TCF12-induced CRC cell stemness was sensitive to niclosamide. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a TCF12-binding long non-coding RNA involved in activation of Wnt/β-catenin signaling pathway and promotion of cancer metastasis. Using RNA immunoprecipitation assay, an interruption of TCF12 binding with MALAT1 was observed in niclosamide-treated CRC cells. Additionally, an E-box pulldown assay revealed that association of TCF12 with Oct-4 on the E-box site of MALAT1 gene promoter was also repressed by niclosamide, which accounted for a reduction of MALAT1 expression but no TCF12 decrease at 24-h niclosamide treatment. In the CRC cells with higher TCF12 expression, the levels of MALAT1, nuclear β-catenin, and the downstream targets cyclin D1 and c-Myc were all increased. These up-regulated expressions were also abrogated after niclosamide treatment. Taken together, our results suggest a mechanism for CRC malignant progression, which also provides a potential therapeutic target.

Keywords: TCF12; MALAT1; niclosamide; Wnt/β-catenin signaling pathway; cancer cell stemnes

摘要 V
Abstract VII
第一章 緒論 1
一、 大腸直腸癌 1
二、 TCF12會導致癌細胞惡化與增加幹細胞性 2
三、 TCF12 與癌症幹細胞 3
四、 長鏈非編碼RNA MALAT1 5
五、 典型Wnt/β-catenin信號途徑 7
六、 Niclosamide具有抗癌幹細胞潛力 9
第二章 研究目標 11
第三章 實驗材料 12
一、 細胞培養 12
二、 藥物與試劑 12
三、 分子量標示物 14
四、 染劑 14
五、 免疫墨點法相關 14
六、 抗體 15
七、 DNA引子 16
八、 實驗儀器 17
第四章 實驗方法 20
一、 細胞培養 20
1. 培養基製備： 20
2. Phosphate-buffered saline與Trypsin-EDTA製備： 20
3. 細胞繼代培養 21
4. 細胞冷凍保存 21
5. TCF12 overexpression和TCF12 knockdown細胞 22
二、 細胞存活率分析 (MTT assay) 22
三、 反轉錄聚合酶連鎖反應 (RT-PCR； Reverse Transcription-Polymerase Chain Reaction): 22
1. 抽取total RNA 22
2. cDNA的製備 (reverse transcription) 23
3. 連鎖聚合反應 (polymerase chain reaction, PCR) 23
4. 瓊脂醣凝膠電泳 24
四、 細胞表面抗原分析 24
五、 細胞球體形成實驗 25
六、 蛋白質的萃取與濃度測定 25
1. 蛋白質的萃取 25
2. 細胞核蛋白與細胞質蛋白的萃取 26
3. 蛋白質濃度的測定 26
七、 免疫墨點法 27
1. SDS-PAGE 蛋白質膠體電泳 27
2. 濕式轉漬 28
八、 E-box pulldown assay 29
第五章 實驗結果 31
第六章 討論 40
第七章 結論 45
第八章 圖表 46
Figure 1: Cytotoxicities of niclosamide and salinomycin in CRC cell lines. 46
Figure 2: TCF12 expression levels determining differential susceptibilities to 5-flurouracil (5-FU), niclosamide, and salinomycin in CRC cells. 47
Figure 3: Suppression of TCF12-associated stemness marker gene expressions by niclosamide. 48
Figure 4: Reduction of TCF12-associated cell-surface stemness marker levels by niclosamide. 49
Figure 5: Inhibition of TCF12-associated cell-spheroid formation abilities by niclosamide. 50
Figure 6: Repression of MALAT1 expression and MALAT1–TCF12 interaction by niclosamide. 51
Figure 7: Disruption of TCF12–Oct-4 interaction by niclosamide on the E-box site of MALAT1 gene promoter. 52
Figure 8: Suppression of TCF12–MALAT1-mediated Wnt/-catenin pathway by niclosamide. 53
Figure 9: Schematic representation of the role of TCF12–MALAT1-mediated stemness. 54
第九章 引用文獻 55
第十章 附錄 62
附錄一、 TCF12 RNAIP定序比對的結果 62
附錄二、 LncRNA調控基因的機制 63
附錄三、 Wnt信號途徑 64
附錄四、 Wnt/β-catenin信號途徑 65

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