臺灣博碩士論文加值系統

慢性B型肝炎病毒 (hepatitis B virus, HBV) 的感染已被證實為導致肝癌的主要危險因子，其中又以HBV中的X蛋白 (HBx) 在肝細胞發展為腫瘤的過程中扮演著關鍵的角色。在先前的肝癌全基因組分析研究中，已鑑定出玻尿酸間接機動性受體 (Receptor for Hyaluronan Mediated Motility, RHAMM) 在肝癌細胞中會過量表現；然而，有關RHAMM對於腫瘤生成及化學抗藥性方面的調控機制仍舊不清楚。因此，在本篇研究中證實HBx會透過PI3K/Akt/C/EBPβ路徑上調RHAMM的表現，並涉及腫瘤轉移與化學治療的結果。此外，HBx的存在也會壓制轉錄因子C/EBPβ結合於RHAMM promoter之活性，進而上調RHAMM的表現。且在細胞移行分析中也發現，抑制RHAMM表現會減緩細胞移行能力；而藥物 (Taxol及Vincristine) 則可反轉其原本抑制細胞生長的能力。此外，透過HBx上調RHAMM表現也會促進藥物所誘導的細胞凋亡。另一方面，在肝細胞瘤病人的資料庫中發現，預後差的病人其RHAMM的mRNA會過量表現。我們的研究結果發現在HBx陽性的肝癌細胞中HBx有正向調節RHAMM的分子機制。這些結果解釋了HBV陽性的肝癌細胞如何進行移行及轉移，同樣地詮釋了RHAMM與標靶治療的關聯性。

Chronic hepatitis B virus (HBV) infection has been identified as a major risk factor in hepatocellular carcinoma (HCC), which is one of the most common cancers worldwide. Evidence suggests that the HBV X protein (HBx) plays a crucial role in HCC development. Genome-wide analysis identified earlier that the receptor for hyaluronan-mediated motility (RHAMM) represents a putative oncogene overexpressed in many human cancers, especially HCC. However, the role of RHAMM in establishment of tumorigenesis and chemoresistance in HCC is still unclear. This study presents our finding that HBx upregulates the expression of RHAMM through PI3K/Akt/C/EBPβ pathway, and its implication in cancer motogenicity and target therapy. First﹐upregulation of RHAMM expression by HBx was discovered in HCC cells through the PI3K/Akt pathway. Furthermore, departure of C/EBPβ from RHAMM gene promoter by HBx was critical for the RHAMM upregulation as revealed by RNAi and ChIP assays. By scratch assay, we also found that knock-down RHAMM partly impaired motogenicity and, surprisingly, partly reversed growth inhibition by mitotoxin drugs (Taxol, Vincristine) in HCC cells. Nevertheless, altered expression of RHAMM by HBx did affect these drugs-induced apoptosis. Using public dataset of HCC patients, we also found enhanced RHAMM mRNA in the patients of poor prognosis. Our findings uncover the molecular mechanism that underlies the upregulation of RHAMM in HBx-positive HCC cells. These results also provide an explanation for cell motogenicity and cancer metastasis of HBV-positive HCC, and the potential relevance of RHAMM in target therapy as well.

指導教授推薦書
口試委員審定書
授權書…………………………………...…….………………………………………………………………iii
致謝……………………………………………………… …….....……….....…………………………………iv
中文摘要…………………………………………………………....…………………………………………v
英文摘要…………………………………………………………….…………………………………… …vi
目錄………………………………………………………………………...............…………………......……vii
第一章 序論………………………………………………………………………………………………1
1-1 HBV基因體構造……………….............................................................................2
1-2 HBx與肝癌關聯性………………………………………………………………3
1-3 與HBx相關的目標基因………….....................................................……….4
1-4 HBx影響細胞存活與凋亡……………..................................................……6
1-5 HBx對細胞週期的影響……………........................................................……8
1-6 RHAMM： an upregulated gene in HCC………………….…..……9
1-7 Oct-1(POU2F1)…………………………………………………………...………11
1-8 C/EBPβ(CCAAT enhancer binding protein (C/EBP)β).........12
第二章 研究目標與實驗設計……………………………………………………………….14
第三章 材料方法……………………………………………………………………………………17
3-1 蛋白純化與抗體製備………………………………………………………. 17
3-2 Recombinant adenovirus的純化與增量…………………………. 17
3-3 細胞培養及HBx過量表現 (overexpression)………………. 18
3-4 細胞轉染 (Transfection)…………………………………………………. 18
3-5 製備recombinant Lentivirus……………………………………………. 19
3-6 建立穩定細胞株 (stable cell line)……………………….…………. 19
3-7 藥物處理…………………………………………...………………………………. 20
3-8 反轉錄聚合酶連鎖反應 (RT-PCR)…………….…………………. 20
3-9 定量聚合酶連鎖反應 (Q-PCR)………….…………….……………. 21
3-10 細胞存活和生長率分析………………………….………………………. 21
3-11 西方墨點法 (Western blotting)………………………………………. 22
3-12 ChIP (Chromatin immunoprecipitation) assay…….…………. 22
3-13 細胞移動分析：刮除法 (wound migration assay)………. 24
第四章 實驗結果……………………………………………………………………………………26
4-1 HBx相關目標基因在HBx-Hep3B與HBx-transgenic mice肝臟組織中上升調節統計結果………………….…………. 26
4-2 在Huh7、Hep3B、HEK293細胞株中，RHAMM確
實會受到HBx的影響而有上升調節的現象細胞培養
及HBx過量表現(overexpression)…………………………….……. 26
4-3 HBx對Oct-1與C/EBPβ轉錄因子結合至RHAMM promoter上之活性的影響………………………………..……………. 27
4-4 HBx對Oct-1，C/EBPβ轉錄因子及RHAMM表現
的影響………………………………………………………………...…...………. 29
4-5 HBx透過C/EBPβ轉錄因子負調節RHAMM的表現………………………………………………………………………..….…………. 29
4-6 HBx促進RHAMM的表現受限於PI3K途徑的抑制……………………………………...………………………………………………. 30
4-7 HBx透過RHAMM誘導HCC腫瘤細胞的移行…….…. 31
4-8 抑制RHAMM表現可反制藥物 (Taxol， vincristine)
抑制細胞生長………….…………………………….………….……………. 33
4-9 HBx可能透過RHAMM表現而加強藥物 (Taxol， vincristine) 抑制細胞生長………………..….….…………….………. 34
4-10 HBx調節RHAMM表現與細胞凋亡的關聯………………. 34
4-11 RHAMM表現與HCC病人預後的關聯………………………. 36
4-12 HBx透過PI3K/Akt訊息傳遞路徑上調RHAMM表
現，進而促進腫瘤細胞轉移…………………………………….……. 36
第五章 討論…………………………………………………………………………………………….38
第六章 圖表…………………………………………………………………………….………………42
參考文獻…………………………………………………………………………………………………….54
附錄……………………………………………………………………………………………………………..61
Table of Contents
Chapter I Introduction........................................................... 1
Chapter II Aims and rationale................................................ 14
Chapter III Materials and methods…………...……..…………. 17
Chapter IV Results………..…………..…………………………. 26
Chapter V Discussions…..…………….....………...……………. 38
Chapter VI Figures...................................................................... 42
References………………………………………………….…… 54
Appendix……...……………….…………………………………...... 61

Andrisani, O.M., and Barnabas, S. (1999). The transcriptional function of the hepatitis B virus X protein and its role in hepatocarcinogenesis (Review). International journal of oncology 15, 373-379.

Antunovic, J., Lemieux, N., and Cromlish, J.A. (1993). The 17 kDa HBx protein encoded by hepatitis B virus interacts with the activation domains of Oct-1, and functions as a coactivator in the activation and repression of a human U6 promoter. Cellular & molecular biology research 39, 463-482.

Casini, P., Nardi, I., and Ori, M. (2010). RHAMM mRNA expression in proliferating and migrating cells of the developing central nervous system. Gene expression patterns : GEP 10, 93-97.

Chami, M., Ferrari, D., Nicotera, P., Paterlini-Brechot, P., and Rizzuto, R. (2003). Caspase-dependent alterations of Ca2+ signaling in the induction of apoptosis by hepatitis B virus X protein. The Journal of biological chemistry 278, 31745-31755.

Cho, I.J., Ki, S.H., Brooks, C., 3rd, and Kim, S.G. (2009). Role of hepatitis B virus X repression of C/EBPbeta activity in the down-regulation of glutathione S-transferase A2 gene: implications in other phase II detoxifying enzyme expression. Xenobiotica; the fate of foreign compounds in biological systems 39, 182-192.

Chung, T.W., Lee, Y.C., and Kim, C.H. (2004). Hepatitis B viral HBx induces matrix metalloproteinase-9 gene expression through activation of ERK and PI-3K/AKT pathways: involvement of invasive potential. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 18, 1123-1125.

Colgrove, R., Simon, G., and Ganem, D. (1989). Transcriptional activation of homologous and heterologous genes by the hepatitis B virus X gene product in cells permissive for viral replication. Journal of virology 63, 4019-4026.
Denhardt, D.T., Mistretta, D., Chambers, A.F., Krishna, S., Porter, J.F., Raghuram, S., and Rittling, S.R. (2003). Transcriptional regulation of osteopontin and the metastatic phenotype: evidence for a Ras-activated enhancer in the human OPN promoter. Clinical & experimental metastasis 20, 77-84.

Diao, J., Garces, R., and Richardson, C.D. (2001). X protein of hepatitis B virus modulates cytokine and growth factor related signal transduction pathways during the course of viral infections and hepatocarcinogenesis. Cytokine & growth factor reviews 12, 189-205.

Dvorchik, I., Demetris, A.J., Geller, D.A., Carr, B.I., Fontes, P., Finkelstein, S.D., Cappella, N.K., and Marsh, J.W. (2007). Prognostic models in hepatocellular carcinoma (HCC) and statistical methodologies behind them. Current pharmaceutical design 13, 1527-1532.

Edin, M.L., Howe, A.K., and Juliano, R.L. (2001). Inhibition of PKA blocks fibroblast migration in response to growth factors. Experimental cell research 270, 214-222.

Fan, R., Li, X., Du, W., Zou, X., Du, R., Zhao, L., Luo, G., Mo, P., Xia, L., Pan, Y., et al. (2011). Adenoviral-mediated RNA interference targeting URG11 inhibits growth of human hepatocellular carcinoma. International journal of cancer Journal international du cancer 128, 2980-2993.

Feitelson, M.A., Duan, L.X., Guo, J., Sun, B., Woo, J., Steensma, K., Horiike, N., and Blumberg, B.S. (1995). X region deletion variants of hepatitis B virus in surface antigen-negative infections and non-A, non-B hepatitis. The Journal of infectious diseases 172, 713-722.

Han, J., Yoo, H.Y., Choi, B.H., and Rho, H.M. (2000). Selective transcriptional regulations in the human liver cell by hepatitis B viral X protein. Biochemical and biophysical research communications 272, 525-530.

He, T.C., Zhou, S., da Costa, L.T., Yu, J., Kinzler, K.W., and Vogelstein, B. (1998). A simplified system for generating recombinant adenoviruses. Proceedings of the National Academy of Sciences of the United States of America 95, 2509-2514.

Jiang, J., Casalegno-Garduno, R., Chen, H., Schmitt, A., Schmitt, M., and Maxwell, C.A. (2010). Multifunctional proteins bridge mitosis with motility and cancer with inflammation and arthritis. TheScientificWorldJournal 10, 1244-1257.

Kidd-Ljunggren, K., Oberg, M., and Kidd, A.H. (1997). Hepatitis B virus X gene 1751 to 1764 mutations: implications for HBeAg status and disease. The Journal of general virology 78 ( Pt 6), 1469-1478.

Kim, H.J., Kim, S.Y., Kim, J., Lee, H., Choi, M., Kim, J.K., and Ahn, J.K. (2008). Hepatitis B virus X protein induces apoptosis by enhancing translocation of Bax to mitochondria. IUBMB life 60, 473-480.

Kim, K.H., and Seong, B.L. (2003). Pro-apoptotic function of HBV X protein is mediated by interaction with c-FLIP and enhancement of death-inducing signal. The EMBO journal 22, 2104-2116.

Kuo, T.C., and Chao, C.C. (2010). Hepatitis B virus X protein prevents apoptosis of hepatocellular carcinoma cells by upregulating SATB1 and HURP expression. Biochemical pharmacology 80, 1093-1102.

Lee, J.S., Heo, J., Libbrecht, L., Chu, I.S., Kaposi-Novak, P., Calvisi, D.F., Mikaelyan, A., Roberts, L.R., Demetris, A.J., Sun, Z., et al. (2006). A novel prognostic subtype of human hepatocellular carcinoma derived from hepatic progenitor cells. Nature medicine 12, 410-416.

Lian, Z., Liu, J., Li, L., Li, X., Clayton, M., Wu, M.C., Wang, H.Y., Arbuthnot, P., Kew, M., Fan, D., et al. (2006). Enhanced cell survival of Hep3B cells by the hepatitis B x antigen effector, URG11, is associated with upregulation of beta-catenin. Hepatology 43, 415-424.

Liu, Q., Chen, J., Liu, L., Zhang, J., Wang, D., Ma, L., He, Y., Liu, Y., Liu, Z., and Wu, J. (2011). The X protein of hepatitis B virus inhibits apoptosis in hepatoma cells through enhancing the methionine adenosyltransferase 2A gene expression and reducing S-adenosylmethionine production. The Journal of biological chemistry 286, 17168-17180.

Lucito, R., and Schneider, R.J. (1992). Hepatitis B virus X protein activates transcription factor NF-kappa B without a requirement for protein kinase C. Journal of virology 66, 983-991.

Ma, J., Sun, T., Park, S., Shen, G., and Liu, J. (2011). The role of hepatitis B virus X protein is related to its differential intracellular localization. Acta biochimica et biophysica Sinica 43, 583-588.

Maxwell, C.A., McCarthy, J., and Turley, E. (2008). Cell-surface and mitotic-spindle RHAMM: moonlighting or dual oncogenic functions? Journal of cell science 121, 925-932.

Mu, Y., Yu, Y., Yue, X., Musarat, I., Gong, R., Zhu, C., Liu, Y., Liu, F., Zhu, Y., and Wu, J. (2011). The X protein of HBV induces HIV-1 long terminal repeat transcription by enhancing the binding of C/EBPbeta and CREB1/2 regulatory proteins to the long terminal repeat of HIV-1. Virus research 156, 81-90.

Ng, M.C., Lam, V.K., Tam, C.H., Chan, A.W., So, W.Y., Ma, R.C., Zee, B.C., Waye, M.M., Mak, W.W., Hu, C., et al. (2010). Association of the POU class 2 homeobox 1 gene (POU2F1) with susceptibility to Type 2 diabetes in Chinese populations. Diabetic medicine : a journal of the British Diabetic Association 27, 1443-1449.

Ng, S.A., and Lee, C. (2011). Hepatitis B virus X gene and hepatocarcinogenesis. Journal of gastroenterology 46, 974-990.

Ortiz-Zapater, E., Pineda, D., Martinez-Bosch, N., Fernandez-Miranda, G., Iglesias, M., Alameda, F., Moreno, M., Eliscovich, C., Eyras, E., Real, F.X., et al. (2012). Key contribution of CPEB4-mediated translational control to cancer progression. Nature medicine 18, 83-90.

Sato, T., Kikkawa, Y., Hiramitsu, T., Yamamoto, T., Goto, N., Matsuoka, S., Nagasaka, T., Watarai, Y., Uchida, K., and Tominaga, Y. (2011). Role of multifunctional cell cycle modulators in advanced secondary hyperparathyroidism. Therapeutic apheresis and dialysis : official peer-reviewed journal of the International Society for Apheresis, the Japanese Society for Apheresis, the Japanese Society for Dialysis Therapy 15 Suppl 1, 26-32.

Satoh, T., Enokido, Y., Aoshima, H., Uchiyama, Y., and Hatanaka, H. (1997). Changes in mitochondrial membrane potential during oxidative stress-induced apoptosis in PC12 cells. Journal of neuroscience research 50, 413-420.

Sebastian, T., and Johnson, P.F. (2006). Stop and go: anti-proliferative and mitogenic functions of the transcription factor C/EBPbeta. Cell cycle 5, 953-957.

Seto, E., Mitchell, P.J., and Yen, T.S. (1990). Transactivation by the hepatitis B virus X protein depends on AP-2 and other transcription factors. Nature 344, 72-74.

Shi, Y., Reiman, T., Li, W., Maxwell, C.A., Sen, S., Pilarski, L., Daniels, T.R., Penichet, M.L., Feldman, R., and Lichtenstein, A. (2007). Targeting aurora kinases as therapy in multiple myeloma. Blood 109, 3915-3921.

Shih, W.L., Kuo, M.L., Chuang, S.E., Cheng, A.L., and Doong, S.L. (2000). Hepatitis B virus X protein inhibits transforming growth factor-beta -induced apoptosis through the activation of phosphatidylinositol 3-kinase pathway. The Journal of biological chemistry 275, 25858-25864.

Sohr, S., and Engeland, K. (2008). RHAMM is differentially expressed in the cell cycle and downregulated by the tumor suppressor p53. Cell cycle 7, 3448-3460.

Szmuness, W. (1978). Hepatocellular carcinoma and the hepatitis B virus: evidence for a causal association. Progress in medical virology Fortschritte der medizinischen Virusforschung Progres en virologie medicale 24, 40-69.
Terradillos, O., de La Coste, A., Pollicino, T., Neuveut, C., Sitterlin, D., Lecoeur, H., Gougeon, M.L., Kahn, A., and Buendia, M.A. (2002). The hepatitis B virus X protein abrogates Bcl-2-mediated protection against Fas apoptosis in the liver. Oncogene 21, 377-386.

Thomson, J.A., Parsons, P.G., and Sturm, R.A. (1993). In vivo and in vitro expression of octamer binding proteins in human melanoma metastases, brain tissue, and fibroblasts. Pigment cell research / sponsored by the European Society for Pigment Cell Research and the International Pigment Cell Society 6, 13-22.

Tu, H., Bonura, C., Giannini, C., Mouly, H., Soussan, P., Kew, M., Paterlini-Brechot, P., Brechot, C., and Kremsdorf, D. (2001). Biological impact of natural COOH-terminal deletions of hepatitis B virus X protein in hepatocellular carcinoma tissues. Cancer research 61, 7803-7810.

Wentz, M.J., Becker, S.A., and Slagle, B.L. (2000). Dissociation of DDB1-binding and transactivation properties of the hepatitis B virus X protein. Virus research 68, 87-92.

Wysocka, J., and Herr, W. (2003). The herpes simplex virus VP16-induced complex: the makings of a regulatory switch. Trends in biochemical sciences 28, 294-304.

Xu, Z., Yen, T.S., Wu, L., Madden, C.R., Tan, W., Slagle, B.L., and Ou, J.H. (2002). Enhancement of hepatitis B virus replication by its X protein in transgenic mice. Journal of virology 76, 2579-2584.

Yue, X., Yang, F., Yang, Y., Mu, Y., Sun, W., Li, W., Xu, D., Wu, J., and Zhu, Y. (2011). Induction of cyclooxygenase-2 expression by hepatitis B virus depends on demethylation-associated recruitment of transcription factors to the promoter. Virology journal 8, 118.

Zhang, X., Dong, N., Yin, L., Cai, N., Ma, H., You, J., Zhang, H., Wang, H., He, R., and Ye, L. (2005). Hepatitis B virus X protein upregulates survivin expression in hepatoma tissues. Journal of medical virology 77, 374-381.
Zhang, X., Zhang, H., and Ye, L. (2006). Effects of hepatitis B virus X protein on the development of liver cancer. The Journal of laboratory and clinical medicine 147, 58-66.

惠韻如，〈探討HBx目標基因在肝細胞中的表現〉，長庚大學，碩士論文，民國99年。