大腸癌在已開發國家中(包括台灣)是消化系統的常見癌症之一。大腸癌的形成原因來自大腸絨毛細胞不斷增生導致。在大腸癌中，ID family基因不僅調節大腸祖細胞的自我更新，也被認為跟腫瘤形成有關，但是ID4 的功能尚未被知曉。在本研究中，我們發現ID4而非其他的ID家族蛋白與病人較差存活率有關，ID4還能調節細胞生長。根據免疫組織染色實驗得知ID4的表現量和未分化型態腫瘤有關。ID4-silenced會調節大腸癌細胞生長，造成G1/S phase arrest和促使細胞凋亡。根據細胞移動路徑分析實驗得知ID4會調節細胞移動能力以及造成Vimentin表現量差異。我們也經由核醣核酸測序結果得知ID4-silenced和癌症增生擴散能力之基因SNAI1, ID1, and BDNF有關。在大腸初始腫瘤中，基因相關性分析結果得知ID4和SNAI1, ID1, and BDNF呈現正相關。在我們研究結果中，ID4在大腸癌扮演一個重要的致癌基因，也有潛力作為大腸癌中之預後指標。

Colon cancer is one of the most common gastrointestinal cancers in developed countries, including Taiwan. The cause of colorectal cancer can be attributed to the uncontrolled proliferation of colon progenitor cells. ID family genes regulate self-renewal of various progenitor cells and have been linked to tumor initiation and progression of cancers including colon cancer; however, the role of ID4 is less understood. In this study, we report that the expression of ID4, but not other ID family genes, is associated with a poor survival outcome in colon cancer, and ID4 regulates proliferation and dissemination of colon cancer cells. Moreover, immunochemistry staining analysis showed that ID4 expression is associated with a poor differentiation status of colon tumors. Knockdown of ID4 expression attenuated growth of colon cancer cells, causing G1/S cell cycle arrest and incremental apoptotic cell ratio. Moreover, time-lapse cell tracking analysis and Q-PCR assays showed that ID4 silencing inhibited migration ability and decreased mesenchymal marker Vimentin expression in colon cancer cells. RNA-seq analysis identified differential expression of several genes in ID4-silenced versus control colon cancer cells, among which SNAI1, ID1, and BDNF have been linked to proliferation and dissemination of cancer cells. Correlation analysis showed that ID4 is positively associated with SNAI1, ID1, and BDNF expression in primary colon tumors. Our findings suggest that ID4 plays a critical oncogenic role in colon cancer and contains the potential to serve as a prognostic marker in colon cancer progression.

Abstract i
中文摘要 iii
致謝 iv
Introduction 1
Colon cancer 1
Inhibitor of DNA binding protein 1
Epithelial-mesenchymal transition (EMT) 2
SNAI family proteins 3
Material and methods 5
Cell culture 5
Immounohistochemistry (IHC) staining 5
Lentiviral Infection 6
Cell tracking analysis 7
Clonogenic assay 7
Immunoblotting 7
Quantitative Real –Time PCR (Q-PCR) 8
RNA sequencing 10
Cell-cycle assay 10
Annexin Ⅴ 10
Statistical Analysis 10
Results 12
ID4 as a prognostic maker associated with poor survival in colon cancer patients 12
ID4 is associated with poor differentiation in colon cancer 12
ID4 regulates cellular growth of colon cancer 13
ID4 regulates cellular migration of colon cancer 14
Genes regulated by ID4 expression 14
Discussion 16
Figure 18
Reference 50

1 Siegel, R. L., Miller, K. D. & Jemal, A. Cancer statistics, 2018. CA Cancer J Clin 68, 7-30, doi:10.3322/caac.21442 (2018).
2 衛生福利部國民健康署. 中華民國105年癌症登記報告.
3 Obrand, D. I. & Gordon, P. H. Incidence and patterns of recurrence following curative resection for colorectal carcinoma. Diseases of the Colon & Rectum 40, 15-24, doi:10.1007/BF02055676 (1997).
4 Benson, A. B., 3rd et al. American Society of Clinical Oncology recommendations on adjuvant chemotherapy for stage II colon cancer. J Clin Oncol 22, 3408-3419, doi:10.1200/JCO.2004.05.063 (2004).
5 Wolmark, N. et al. The benefit of leucovorin-modulated fluorouracil as postoperative adjuvant therapy for primary colon cancer: results from National Surgical Adjuvant Breast and Bowel Project protocol C-03. Journal of Clinical Oncology 11, 1879-1887, doi:10.1200/JCO.1993.11.10.1879 (1993).
6 Moertel, C. G. et al. Levamisole and Fluorouracil for Adjuvant Therapy of Resected Colon Carcinoma. New England Journal of Medicine 322, 352-358, doi:10.1056/NEJM199002083220602 (1990).
7 Umetani, N. et al. Epigenetic inactivation of ID4 in colorectal carcinomas correlates with poor differentiation and unfavorable prognosis.
8 Kreider, B. L., Benezra, R., Rovera, G. & Kadesch, T. Inhibition of myeloid differentiation by the helix-loop-helix protein Id. Science 255, 1700, doi:10.1126/science.1372755 (1992).
9 Patel, D., Morton, D. J., Carey, J., Havrda, M. C. & Chaudhary, J. Inhibitor of differentiation 4 (ID4): From development to cancer. Biochim Biophys Acta 1855, 92-103, doi:10.1016/j.bbcan.2014.12.002 (2015).
10 Zebedee, Z. & Hara, E. Id proteins in cell cycle control and cellular senescence. Oncogene 20, 8317-8325, doi:10.1038/sj.onc.1205092 (2001).
11 Peverali, F. A. et al. Regulation of G1 progression by E2A and Id helix-loop-helix proteins.
12 Prabhu, S., Ignatova A Fau - Park, S. T., Park St Fau - Sun, X. H. & Sun, X. H. Regulation of the expression of cyclin-dependent kinase inhibitor p21 by E2A and Id proteins.
13 Yun, K., Mantani, A., Garel, S., Rubenstein, J. & Israel, M. A. Id4 regulates neural progenitor proliferation and differentiation in vivo. Development 131, 5441, doi:10.1242/dev.01430 (2004).
14 Barone, M. V., Pepperkok, R., Peverali, F. A. & Philipson, L. Id proteins control growth induction in mammalian cells. Proc Natl Acad Sci U S A 91, 4985-4988, doi:10.1073/pnas.91.11.4985 (1994).
15 Hara, E. et al. Id-related genes encoding helix-loop-helix proteins are required for G1 progression and are repressed in senescent human fibroblasts.
16 O'Brien, C. A. et al. ID1 and ID3 regulate the self-renewal capacity of human colon cancer-initiating cells through p21. Cancer Cell 21, 777-792, doi:10.1016/j.ccr.2012.04.036 (2012).
17 Shan, L., Yu M Fau - Qiu, C., Qiu C Fau - Snyderwine, E. G. & Snyderwine, E. G. Id4 regulates mammary epithelial cell growth and differentiation and is overexpressed in rat mammary gland carcinomas.
18 Kalluri, R. & Neilson, E. G. Epithelial-mesenchymal transition and its implications for fibrosis. Journal of Clinical Investigation 112, 1776-1784, doi:10.1172/jci200320530 (2003).
19 Pena, C. et al. E-cadherin and vitamin D receptor regulation by SNAIL and ZEB1 in colon cancer: clinicopathological correlations. Hum Mol Genet 14, 3361-3370, doi:10.1093/hmg/ddi366 (2005).
20 Batlle, E. et al. The transcription factor Snail is a repressor of E-cadherin gene expression in epithelial tumour cells. Nature Cell Biology 2, 84-89, doi:10.1038/35000034 (2000).
21 Cano, A. et al. The transcription factor Snail controls epithelial–mesenchymal transitions by repressing E-cadherin expression. Nature Cell Biology 2, 76-83, doi:10.1038/35000025 (2000).
22 Thiery, J. P. Epithelial–mesenchymal transitions in tumour progression. Nature Reviews Cancer 2, 442-454, doi:10.1038/nrc822 (2002).
23 Peinado, H., Olmeda, D. & Cano, A. Snail, Zeb and bHLH factors in tumour progression: an alliance against the epithelial phenotype? Nature Reviews Cancer 7, 415, doi:10.1038/nrc2131 (2007).
24 Kim, Y. H. et al. TWIST1 and SNAI1 as markers of poor prognosis in human colorectal cancer are associated with the expression of ALDH1 and TGF-beta1. Oncol Rep 31, 1380-1388, doi:10.3892/or.2014.2970 (2014).
25 Jorda, M. et al. Id-1 is induced in MDCK epithelial cells by activated Erk/MAPK pathway in response to expression of the Snail and E47 transcription factors. Exp Cell Res 313, 2389-2403, doi:10.1016/j.yexcr.2007.04.001 (2007).
26 Hajra, K. M., Chen, D. Y. S. & Fearon, E. R. The SLUG Zinc-Finger Protein Represses E-Cadherin in Breast Cancer. Cancer Research 62, 1613 (2002).