Hairy/Enhancer of split-related with YRPW-like motif (HEY)的蛋白質家族，是一個轉錄調控抑制因子，同時調控Notch下游訊息傳遞路徑的因子。在之前的文獻指出HeyL基因與心血管系統發育中扮演極重要的角色，在HeyL失去正常作用功能時，可能會導致vascular specification、septation以及valve formation等相關的缺陷症狀。在本篇研究中，我們針對肝癌病患的檢體去探討BMP4相關的下游基因，發現在大量表達BMP4的肝癌病患檢體中，HeyL的會表現被抑制。所以我們假設HeyL可能在肝癌腫瘤演變以及腫瘤生長過程中扮演重要角色。同時我們也想要建立出HeyL相關的訊息調控路徑作為治療肝癌的基礎，期望夠釐清下游調控腫瘤增生和腫瘤轉移的分子機制。更進一步了解，HeyL相關的功能以及其訊息調控傳遞路徑與在肝癌腫瘤的生物角色，且提供另一種更佳的治療策略，也能作為協助臨床診斷的依據以及肝癌發生的預測指標。

The Hairy/Enhancer of split-related with YRPW-like motif (HEY) family of proteins were first identified as transcriptional repressors and were downstream effectors of Notch and BMP pathways. Many studies indicated that HeyL gene plays an important role in the development of the cardiovascular system and loss of HeyL function causes defects in vascular specification, septation and valve formation. In this study, we screened the alterations of BMP4 downstream effectors/genes expression in primary HCC samples, and identified expression of HeyL was decreased in HCC. We also found that HeyL was significantly reduced in HCC cell lines after BMP4 treatment, so we hypothesize that HeyL may serve a tumor suppressor in HCC carcinogenesis. In this study, we also identified HeyL binding networks to examine its major binding partners involved in regulated tumor cell proliferation or tumor metastasis. Furthermore, the understanding of the modes of HeyL function and its biological roles in HCC could undoubtedly provide ideal nodal points of therapeutic intervention in addition to aiding in the clinical diagnosis and prognosis of this devastating disease.

中文摘要 i

Abstract ii

Abbreviations iii

Content iv

I.Introductions - 1 -

Hepatocellular carcinoma (HCC) - 1 -

The Hairy/Enhancer of split-related with YRPW-like motif family (HEY family) - 2 -

TGF-β signaling pathway and its role in tumor progression - 4 -

The relationship of tumor genesis and epigenetic regulations - 5 -

Liquid chromatography–mass spectrometry (LC-MS) - 6 -

II.Material and Methods - 7 -

RNA isolation, cDNA synthesis and Quantitative real-time PCR - 7 -

Hematoxylin and Eosin staining - 8 -

Immunohistochemistry staining - 9 -

Genomic DNA isolation and Bisulfide treatment - 10 -

Methylation-Specific PCR - 10 -

Cell culture - 11 -

HeyL overexpression by lipofectamine2000 transfection - 12 -

MTT assay - 12 -

Luciferase reporter assay - 13 -

Western Blotting - 14 -

Wound healing assay - 15 -

Protein co-immunoprecipitation - 16 -

In-gel tryptic digestion and protein identification by HPLC-Mass Spectrometry (LC-MS) - 17 -

III.Results - 18 -

HeyL is down-regulated in HCC patient tissues. - 18 -

HeyL promoter is hyper-methylated in HCC patient tissues and malignant cell lines. - 18 -

HeyL overexpression in HCC cell line attenuates its proliferation and migration. - 19 -

HeyL may relate to TGF-Smad signaling pathway in HCC cell line. - 20 -

Analyze of HeyL-related proteins in HCC cell line by Immunoprecipitation-HPLC-Mass Spectrometry. - 21 -

IV.Discussion - 22 -

V.Figures and tables - 25 -

Figure 1. The downregulation of HeyL mRNA expression of in HCC patient. - 26 -

Figure 2. The decreased protein expression of HeyL in human liver cancer. - 27 -

Figure 3. The increased methylation level of HeyL promoter in human HCC samples. - 28 -

Figure 4. Overexpression of HeyL inhibits HCC cell line’s proliferation and migration of HCC cells. - 29 -

Figure 5. HeyL overexpression in SK-Hep1 cell line decreased 3TP-Luc reporter activity. - 31 -

Figure 6. Identification HeyL interacting proteins by IP-LC-MS analysis. - 32 -

Table 1. Identification of proteins interacting with HeyL from Huh7 control cells by IP- LC-MS/MS analysis. - 36 -

Table 2. Identification of proteins interacting with HeyL from HeyL overexpressing Huh7 cells by IP-LC-MS/MS. - 41 -

Table 3. The list of identified proteins interacting with HeyL in Huh7 cell line. - 45 -

VI.Reference - 51 -

Baylin, S.B., and Ohm, J.E. (2006). Epigenetic gene silencing in cancer - a mechanism for early oncogenic pathway addiction? Nature reviews Cancer 6, 107-116.

Belandia, B., Powell, S.M., Garcia-Pedrero, J.M., Walker, M.M., Bevan, C.L., and Parker, M.G. (2005). Hey1, a mediator of notch signaling, is an androgen receptor corepressor. Molecular and cellular biology 25, 1425-1436.

Carpenter, B., MacKay, C., Alnabulsi, A., MacKay, M., Telfer, C., Melvin, W.T., and Murray, G.I. (2006). The roles of heterogeneous nuclear ribonucleoproteins in tumour development and progression. Biochimica et biophysica acta 1765, 85-100.

Colland, F., Jacq, X., Trouplin, V., Mougin, C., Groizeleau, C., Hamburger, A., Meil, A., Wojcik, J., Legrain, P., and Gauthier, J.M. (2004). Functional proteomics mapping of a human signaling pathway. Genome research 14, 1324-1332.

Cui, H., Wu, F., Sun, Y., Fan, G., and Wang, Q. (2010). Up-regulation and subcellular localization of hnRNP A2/B1 in the development of hepatocellular carcinoma. BMC cancer 10, 356.

Davis, R.L., and Turner, D.L. (2001). Vertebrate hairy and Enhancer of split related proteins: transcriptional repressors regulating cellular differentiation and embryonic patterning. Oncogene 20, 8342-8357.

Feng, X.H., and Derynck, R. (2005). Specificity and versatility in tgf-beta signaling through Smads. Annual review of cell and developmental biology 21, 659-693.

Fischer, A., Schumacher, N., Maier, M., Sendtner, M., and Gessler, M. (2004). The Notch target genes Hey1 and Hey2 are required for embryonic vascular development. Genes & development 18, 901-911.

Fischer, A., Steidl, C., Wagner, T.U., Lang, E., Jakob, P.M., Friedl, P., Knobeloch, K.P., and Gessler, M. (2007). Combined loss of Hey1 and HeyL causes congenital heart defects because of impaired epithelial to mesenchymal transition. Circulation research 100, 856-863.

Gupta, G.P., and Massague, J. (2006). Cancer metastasis: building a framework. Cell 127, 679-695.

Han, S.P., Tang, Y.H., and Smith, R. (2010). Functional diversity of the hnRNPs: past, present and perspectives. The Biochemical journal 430, 379-392.

Hanahan, D., and Weinberg, R.A. (2000). The hallmarks of cancer. Cell 100, 57-70.

Hanahan, D., and Weinberg, R.A. (2011). Hallmarks of cancer: the next generation. Cell 144, 646-674.

Herman, J.G., and Baylin, S.B. (2003). Gene silencing in cancer in association with promoter hypermethylation. The New England journal of medicine 349, 2042-2054.

Hoshida, Y., Villanueva, A., Kobayashi, M., Peix, J., Chiang, D.Y., Camargo, A., Gupta, S., Moore, J., Wrobel, M.J., Lerner, J., et al. (2008). Gene expression in fixed tissues and outcome in hepatocellular carcinoma. The New England journal of medicine 359, 1995-2004.

Iso, T., Kedes, L., and Hamamori, Y. (2003). HES and HERP families: multiple effectors of the Notch signaling pathway. Journal of cellular physiology 194, 237-255.

Kim, N.W., Piatyszek, M.A., Prowse, K.R., Harley, C.B., West, M.D., Ho, P.L., Coviello, G.M., Wright, W.E., Weinrich, S.L., and Shay, J.W. (1994). Specific association of human telomerase activity with immortal cells and cancer. Science 266, 2011-2015.

Klambt, C., Knust, E., Tietze, K., and Campos-Ortega, J.A. (1989). Closely related transcripts encoded by the neurogenic gene complex enhancer of split of Drosophila melanogaster. The EMBO journal 8, 203-210.

LaBranche, H., Dupuis, S., Ben-David, Y., Bani, M.R., Wellinger, R.J., and Chabot, B. (1998). Telomere elongation by hnRNP A1 and a derivative that interacts with telomeric repeats and telomerase. Nature genetics 19, 199-202.

Lavery, D.N., Villaronga, M.A., Walker, M.M., Patel, A., Belandia, B., and Bevan, C.L. (2011). Repression of androgen receptor activity by HEYL, a third member of the Hairy/Enhancer-of-split-related family of Notch effectors. The Journal of biological chemistry 286, 17796-17808.

Lecourtois, M., and Schweisguth, F. (1995). The neurogenic suppressor of hairless DNA-binding protein mediates the transcriptional activation of the enhancer of split complex genes triggered by Notch signaling. Genes & development 9, 2598-2608.

Leimeister, C., Externbrink, A., Klamt, B., and Gessler, M. (1999). Hey genes: a novel subfamily of hairy- and Enhancer of split related genes specifically expressed during mouse embryogenesis. Mechanisms of development 85, 173-177.

Llovet, J.M., and Bruix, J. (2008a). Molecular targeted therapies in hepatocellular carcinoma. Hepatology 48, 1312-1327.

Llovet, J.M., and Bruix, J. (2008b). Novel advancements in the management of hepatocellular carcinoma in 2008. Journal of hepatology 48 Suppl 1, S20-37.

Llovet, J.M., Burroughs, A., and Bruix, J. (2003). Hepatocellular carcinoma. Lancet 362, 1907-1917.

Ma, Y.L., Peng, J.Y., Zhang, P., Huang, L., Liu, W.J., Shen, T.Y., Chen, H.Q., Zhou, Y.K., Zhang, M., Chu, Z.X., et al. (2009). Heterogeneous nuclear ribonucleoprotein A1 is identified as a potential biomarker for colorectal cancer based on differential proteomics technology. Journal of proteome research 8, 4525-4535.

Massague, J. (2008). TGFbeta in Cancer. Cell 134, 215-230.

Matta, A., Tripathi, S.C., DeSouza, L.V., Grigull, J., Kaur, J., Chauhan, S.S., Srivastava, A., Thakar, A., Shukla, N.K., Duggal, R., et al. (2009). Heterogeneous ribonucleoprotein K is a marker of oral leukoplakia and correlates with poor prognosis of squamous cell carcinoma. International journal of cancer Journal international du cancer 125, 1398-1406.

Meeker, A.K., Hicks, J.L., Platz, E.A., March, G.E., Bennett, C.J., Delannoy, M.J., and De Marzo, A.M. (2002). Telomere shortening is an early somatic DNA alteration in human prostate tumorigenesis. Cancer research 62, 6405-6409.

Meindl-Beinker, N.M., Matsuzaki, K., and Dooley, S. (2012). TGF-beta signaling in onset and progression of hepatocellular carcinoma. Digestive diseases 30, 514-523.

Mishra, L., Banker, T., Murray, J., Byers, S., Thenappan, A., He, A.R., Shetty, K., Johnson, L., and Reddy, E.P. (2009). Liver stem cells and hepatocellular carcinoma. Hepatology 49, 318-329.

Padua, D., and Massague, J. (2009). Roles of TGFbeta in metastasis. Cell research 19, 89-102.

Parkin, D.M., Bray, F., Ferlay, J., and Pisani, P. (2005). Global cancer statistics, 2002. CA: a cancer journal for clinicians 55, 74-108.

Pio, R., Zudaire, I., Pino, I., Castano, Z., Zabalegui, N., Vicent, S., Garcia-Amigot, F., Odero, M.D., Lozano, M.D., Garcia-Foncillas, J., et al. (2004). Alpha CP-4, encoded by a putative tumor suppressor gene at 3p21, but not its alternative splice variant alpha CP-4a, is underexpressed in lung cancer. Cancer research 64, 4171-4179.

Ross, S., and Hill, C.S. (2008). How the Smads regulate transcription. The international journal of biochemistry & cell biology 40, 383-408.

Roy, M., Pear, W.S., and Aster, J.C. (2007). The multifaceted role of Notch in cancer. Current opinion in genetics & development 17, 52-59.

Shimoda, M., Takahashi, M., Yoshimoto, T., Kono, T., Ikai, I., and Kubo, H. (2006). A homeobox protein, prox1, is involved in the differentiation, proliferation, and prognosis in hepatocellular carcinoma. Clinical cancer research : an official journal of the American Association for Cancer Research 12, 6005-6011.

Tauler, J., Zudaire, E., Liu, H., Shih, J., and Mulshine, J.L. (2010). hnRNP A2/B1 modulates epithelial-mesenchymal transition in lung cancer cell lines. Cancer research 70, 7137-7147.

Ushigome, M., Ubagai, T., Fukuda, H., Tsuchiya, N., Sugimura, T., Takatsuka, J., and Nakagama, H. (2005). Up-regulation of hnRNP A1 gene in sporadic human colorectal cancers. International journal of oncology 26, 635-640.

Villaronga, M.A., Bevan, C.L., and Belandia, B. (2008). Notch signaling: a potential therapeutic target in prostate cancer. Current cancer drug targets 8, 566-580.

Yang, L., Inokuchi, S., Roh, Y.S., Song, J., Loomba, R., Park, E.J., and Seki, E. (2013). Transforming Growth Factor-beta Signaling in Hepatocytes Promotes Hepatic Fibrosis and Carcinogenesis in Mice With Hepatocyte-Specific Deletion of TAK1. Gastroenterology 144, 1042-1054 e1044.

Zhou, J., Nong, L., Wloch, M., Cantor, A., Mulshine, J.L., and Tockman, M.S. (2001). Expression of early lung cancer detection marker: hnRNP-A2/B1 and its relation to microsatellite alteration in non-small cell lung cancer. Lung cancer 34, 341-350.