臺灣博碩士論文加值系統

抑癌基因異常的DNA高度甲基化會抑制該基因的表現，並且促使癌症的形成。由於抑癌基因的甲基化程度可以當作腫瘤進展的一種生物標誌，我們利用甲基專ㄧ之高解析度溶解曲線 (methylation specific high resolution melting, MS-HRM) PCR技術去偵測鼻咽癌檢體中抑癌基因的甲基化程度。之前我們發現一個高度甲基化基因在鼻咽癌檢體中，此基因主要為調控體軸發展的轉錄因子，我們以亞硫酸鹽定序(bisulfite sequencing) 分析鼻咽癌細胞株與鼻咽癌檢體，發現Gene A是被高度甲基化(65~95%)；而鼻咽癌鄰近正常組織中，Gene A甲基程度卻較低(8~65%)。因此我們以MS-HRM分析相同的鼻咽癌細胞株以及鼻咽癌檢體，並比較兩種方法所得的結果發現，MS-HRM和亞硫酸鹽定序分析結果一致。Gene A只有在鼻咽癌檢體和鼻咽癌細胞株均呈現高度甲基化(34~84%)，但是在鼻咽癌周邊正常組織和正常人的白血球中Gene A基因則是甲基程度較低(1~12%)。另外，MS-HRM 分析相較於亞硫酸鹽定序在操作上更快速且經濟。由於血液中EB病毒量本身被視為腫瘤預後標誌，為了進一步了解Gene A甲基程度與已知的標誌的關聯性，我們從5位鼻咽癌病人在不同時間裡取得的27個血漿樣品中分離游離DNA，並分別測定EB病毒的copy number和Gene A的甲基化程度，結果顯示兩者有正相關，這表示血漿中EB病毒量的多寡可反應腫瘤中Gene A的甲基程度。因此我們發展以MS-HRM技術可快速、準確偵測檢體中Gene A基因的甲基程度，作為鼻咽癌的生物指標。

Aberrant DNA hypermethylation of tumor suppressor genes (TSGs) inhibits gene expression and causes tumor progression. Since TSGs hypermethylation often used as biomarkers reflecting tumor progression, we used methylation specific high resolution melting (MS-HRM) PCR-based analysis to rapidly detect DNA methyation percentage of aberrant hypermethylated genes in nasopharyngeal carcinoma (NPC). We previously identified a novel hypermethylated gene, Gene A, in NPC biopsies. Gene A is a transcription factor which regulates body axes development. According to our bisulfite sequencing data , Gene A was hypermethylated in NPC cell lines and NPC tumor biopsies (65~95%), however, Gene A gene of adjacent normal tissues and normal individuals’ white blood cells was hypomethylated (8~65%). To test whether MS-HRM has comparable results as bisulfite sequencing, we performed MS-HRM using the same samples. Indeed, MS-HRM had consistent results as bisulfite sequencing. Besides, MS-HRM is a more rapid and economic analysis when compared with bisulfite sequencing . Since EBV copy number is considered as prognostic marker for NPC, we isolated 27 cell-free DNA samples from 5 NPC patients’ plasma at difference time. We further detected EBV copy number and Gene A methylation, respectively. Our results indicated that EBV copy number was positively correlated with Gene A methylation . Hence, the copy number of EBV in plasma could reflect Gene A methylation level in tumor. Taken together, we have developed a rapid and accurate MS-HRM method to detect Gene A hypermethylation; we may use this gene as a biomarker for NPC.

目錄
指導教授推薦書 i
口試委員會審定書 ii
授權書 iii
誌謝 iv
中文摘要 vi
英文摘要 vii
目錄 viii
第一章、前言 1
1.1鼻咽癌(Nasopharyngeal Carcinoma, NPC) 1
1.2 EBV病毒(Epstein-Barr virus) 1
1.3 表觀遺傳(Epigenetics)和DNA甲基化 3
1.4 抑癌基因(Tumour Suppressor Genes, TSGs) 6
1.5 甲基敏感性高解析度溶解曲線(Methylation-Sensitive High Resolution Melting, MS-HRM) 8
1.6 研究動機與目標 11
第二章、材料與方法 13
2.1 Methylated (M)和Unmethylated (U)質體建構 13
2.2 亞硫酸鹽處理 14
2.3 細胞培養 14
2.4 鼻咽癌檢體 15
2.5 萃取白血球、血漿DNA 15
2.6 萃取細胞DNA 16
2.7 高解析度溶解曲線 17
第三章、結果 18
3.1 Gene A啟動子區域的CpG islands分佈情形 18
3.2 建立用於MS-HRM偵測Gene A基因啟動子的標準曲線 19
3.3 利用MS-HRM分析經過亞硫酸鹽定序的質體 19
3.4 利用MS-HRM分析鼻咽癌病人的檢體和周邊正常鼻咽組織中Gene A的甲基化程度 20
3.5 利用MS-HRM和亞硫酸鹽定序分析鼻咽癌細胞株的甲基化程度 21
3.6 連結Gene A甲基化和EBV的copy number 22
3.7 比較MS-HRM和亞硫酸鹽定序 22
3.8 MS-HRM應用於偵測鼻咽癌中其他抑癌基因的甲基程度 24
第四章、討論 25
參考文獻 29
圖、表附錄 38

參考文獻
Agathanggelou, A., Dallol, A., Zochbauer-Muller, S., Morrissey, C., Honorio, S., Hesson, L., Martinsson, T., Fong, K.M., Kuo, M.J., Yuen, P.W., et al. (2003). Epigenetic inactivation of the candidate 3p21.3 suppressor gene BLU in human cancers. Oncogene 22, 1580-1588.
Ayadi, W., Karray-Hakim, H., Khabir, A., Feki, L., Charfi, S., Boudawara, T., Ghorbel, A., Daoud, J., Frikha, M., Busson, P., et al. (2008). Aberrant methylation of p16, DLEC1, BLU and E-cadherin gene promoters in nasopharyngeal carcinoma biopsies from Tunisian patients. Anticancer Res 28, 2161-2167.
Baumforth, K.R., Young, L.S., Flavell, K.J., Constandinou, C., and Murray, P.G. (1999). The Epstein-Barr virus and its association with human cancers. Mol Pathol 52, 307-322.
Baylin, S.B., Esteller, M., Rountree, M.R., Bachman, K.E., Schuebel, K., and Herman, J.G. (2001). Aberrant patterns of DNA methylation, chromatin formation and gene expression in cancer. Hum Mol Genet 10, 687-692.
Bernards, A. (2003). GAPs galore! A survey of putative Ras superfamily GTPase activating proteins in man and Drosophila. Biochim Biophys Acta 1603, 47-82.
Bernstein, B.E., Meissner, A., and Lander, E.S. (2007). The mammalian epigenome. Cell 128, 669-681.
Bird, A. (2002). DNA methylation patterns and epigenetic memory. Genes Dev 16, 6-21.
Cedar, H., and Bergman, Y. (2009). Linking DNA methylation and histone modification: patterns and paradigms. Nat Rev Genet 10, 295-304.
Chang, Y.S., Shih, L.Y., Peng, C.L., Chen, S.H., and Liu, S.T. (1990). Characterization of two newly established EBV-containing lymphoblastoid cell lines from patients with myeloid leukemias. Leuk Res 14, 309-320.
Chen, T., and Li, E. (2006). Establishment and maintenance of DNA methylation patterns in mammals. Curr Top Microbiol Immunol 301, 179-201.
Cheng, X., and Blumenthal, R.M. (2008). Mammalian DNA methyltransferases: a structural perspective. Structure 16, 341-350.
Cheung, S.T., Huang, D.P., Hui, A.B., Lo, K.W., Ko, C.W., Tsang, Y.S., Wong, N., Whitney, B.M., and Lee, J.C. (1999). Nasopharyngeal carcinoma cell line (C666-1) consistently harbouring Epstein-Barr virus. Int J Cancer 83, 121-126.
Chou, J., Lin, Y.C., Kim, J., You, L., Xu, Z., He, B., and Jablons, D.M. (2008). Nasopharyngeal carcinoma--review of the molecular mechanisms of tumorigenesis. Head Neck 30, 946-963.
Comb, M., and Goodman, H.M. (1990). CpG methylation inhibits proenkephalin gene expression and binding of the transcription factor AP-2. Nucleic Acids Res 18, 3975-3982.
Daniel, F.I., Cherubini, K., Yurgel, L.S., de Figueiredo, M.A., and Salum, F.G. (2011). The role of epigenetic transcription repression and DNA methyltransferases in cancer. Cancer 117, 677-687.
Durkin, M.E., Yuan, B.Z., Zhou, X., Zimonjic, D.B., Lowy, D.R., Thorgeirsson, S.S., and Popescu, N.C. (2007). DLC-1:a Rho GTPase-activating protein and tumour suppressor. J Cell Mol Med 11, 1185-1207.
Epstein, M.A., Achong, B.G., and Barr, Y.M. (1964). Virus Particles in Cultured Lymphoblasts from Burkitt's Lymphoma. Lancet 1, 702-703.
Fahraeus, R., Fu, H.L., Ernberg, I., Finke, J., Rowe, M., Klein, G., Falk, K., Nilsson, E., Yadav, M., Busson, P., et al. (1988). Expression of Epstein-Barr virus-encoded proteins in nasopharyngeal carcinoma. Int J Cancer 42, 329-338.
Feinberg, A.P., and Tycko, B. (2004). The history of cancer epigenetics. Nat Rev Cancer 4, 143-153.
Goldsmith, D.B., West, T.M., and Morton, R. (2002). HLA associations with nasopharyngeal carcinoma in Southern Chinese: a meta-analysis. Clin Otolaryngol Allied Sci 27, 61-67.
Goll, M.G., Kirpekar, F., Maggert, K.A., Yoder, J.A., Hsieh, C.L., Zhang, X., Golic, K.G., Jacobsen, S.E., and Bestor, T.H. (2006). Methylation of tRNAAsp by the DNA methyltransferase homolog Dnmt2. Science 311, 395-398.
Huang, D.P., Ho, J.H., Poon, Y.F., Chew, E.C., Saw, D., Lui, M., Li, C.L., Mak, L.S., Lai, S.H., and Lau, W.H. (1980). Establishment of a cell line (NPC/HK1) from a differentiated squamous carcinoma of the nasopharynx. Int J Cancer 26, 127-132.
Hutajulu, S.H., Indrasari, S.R., Indrawati, L.P., Harijadi, A., Duin, S., Haryana, S.M., Steenbergen, R.D., Greijer, A.E., and Middeldorp, J.M. (2011). Epigenetic markers for early detection of nasopharyngeal carcinoma in a high risk population. Mol Cancer 10, 48.
Jones, P.A., and Laird, P.W. (1999). Cancer epigenetics comes of age. Nat Genet 21, 163-167.
Khabir, A., Karray, H., Rodriguez, S., Rose, M., Daoud, J., Frikha, M., Boudawara, T., Middeldorp, J., Jlidi, R., and Busson, P. (2005). EBV latent membrane protein 1 abundance correlates with patient age but not with metastatic behavior in north African nasopharyngeal carcinomas. Virol J 2, 39.
Kim, T.Y., Jong, H.S., Song, S.H., Dimtchev, A., Jeong, S.J., Lee, J.W., Kim, N.K., Jung, M., and Bang, Y.J. (2003). Transcriptional silencing of the DLC-1 tumor suppressor gene by epigenetic mechanism in gastric cancer cells. Oncogene 22, 3943-3951.
Klein, G. (1987). The approaching era of the tumor suppressor genes. Science 238, 1539-1545.
Lemons, D., and McGinnis, W. (2006). Genomic evolution of Hox gene clusters. Science 313, 1918-1922.
Leonhardt, H., Page, A.W., Weier, H.U., and Bestor, T.H. (1992). A targeting sequence directs DNA methyltransferase to sites of DNA replication in mammalian nuclei. Cell 71, 865-873.
Liao, S.K., Perng, Y.P., Shen, Y.C., Chung, P.J., Chang, Y.S., and Wang, C.H. (1998). Chromosomal abnormalities of a new nasopharyngeal carcinoma cell line (NPC-BM1) derived from a bone marrow metastatic lesion. Cancer Genet Cytogenet 103, 52-58.
Lin, C.T., Chan, W.Y., Chen, W., Huang, H.M., Wu, H.C., Hsu, M.M., Chuang, S.M., and Wang, C.C. (1993). Characterization of seven newly established nasopharyngeal carcinoma cell lines. Lab Invest 68, 716-727.
Lin, C.T., Wong, C.I., Chan, W.Y., Tzung, K.W., Ho, J.K., Hsu, M.M., and Chuang, S.M. (1990). Establishment and characterization of two nasopharyngeal carcinoma cell lines. Lab Invest 62, 713-724.
Liu, X.Q., Chen, H.K., Zhang, X.S., Pan, Z.G., Li, A., Feng, Q.S., Long, Q.X., Wang, X.Z., and Zeng, Y.X. (2003). Alterations of BLU, a candidate tumor suppressor gene on chromosome 3p21.3, in human nasopharyngeal carcinoma. Int J Cancer 106, 60-65.
Lo, K.W., To, K.F., and Huang, D.P. (2004). Focus on nasopharyngeal carcinoma. Cancer Cell 5, 423-428.
Lo, Y.M., Chan, A.T., Chan, L.Y., Leung, S.F., Lam, C.W., Huang, D.P., and Johnson, P.J. (2000). Molecular prognostication of nasopharyngeal carcinoma by quantitative analysis of circulating Epstein-Barr virus DNA. Cancer Res 60, 6878-6881.
Lo, Y.M., Chan, L.Y., Chan, A.T., Leung, S.F., Lo, K.W., Zhang, J., Lee, J.C., Hjelm, N.M., Johnson, P.J., and Huang, D.P. (1999). Quantitative and temporal correlation between circulating cell-free Epstein-Barr virus DNA and tumor recurrence in nasopharyngeal carcinoma. Cancer Res 59, 5452-5455.
Low, J.S., Tao, Q., Ng, K.M., Goh, H.K., Shu, X.S., Woo, W.L., Ambinder, R.F., Srivastava, G., Shamay, M., Chan, A.T., et al. (2011). A novel isoform of the 8p22 tumor suppressor gene DLC1 suppresses tumor growth and is frequently silenced in multiple common tumors. Oncogene 30, 1923-1935.
Magrath, I.T. (1991). African Burkitt's lymphoma. History, biology, clinical features, and treatment. Am J Pediatr Hematol Oncol 13, 222-246.
Martin, G.S. (2003). Cell signaling and cancer. Cancer Cell 4, 167-174.
Miranda, T.B., and Jones, P.A. (2007). DNA methylation: the nuts and bolts of repression. J Cell Physiol 213, 384-390.
Moon, S.Y., and Zheng, Y. (2003). Rho GTPase-activating proteins in cell regulation. Trends Cell Biol 13, 13-22.
Okano, M., Bell, D.W., Haber, D.A., and Li, E. (1999). DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99, 247-257.
Pang, J.C., Chang, Q., Chung, Y.F., Teo, J.G., Poon, W.S., Zhou, L.F., Kong, X., and Ng, H.K. (2005). Epigenetic inactivation of DLC-1 in supratentorial primitive neuroectodermal tumor. Hum Pathol 36, 36-43.
Peng, D., Ren, C.P., Yi, H.M., Zhou, L., Yang, X.Y., Li, H., and Yao, K.T. (2006). Genetic and epigenetic alterations of DLC-1, a candidate tumor suppressor gene, in nasopharyngeal carcinoma. Acta Biochim Biophys Sin (Shanghai) 38, 349-355.
Qiu, G.H., Tan, L.K., Loh, K.S., Lim, C.Y., Srivastava, G., Tsai, S.T., Tsao, S.W., and Tao, Q. (2004). The candidate tumor suppressor gene BLU, located at the commonly deleted region 3p21.3, is an E2F-regulated, stress-responsive gene and inactivated by both epigenetic and genetic mechanisms in nasopharyngeal carcinoma. Oncogene 23, 4793-4806.
Robert, M.F., Morin, S., Beaulieu, N., Gauthier, F., Chute, I.C., Barsalou, A., and MacLeod, A.R. (2003). DNMT1 is required to maintain CpG methylation and aberrant gene silencing in human cancer cells. Nat Genet 33, 61-65.
Robertson, K.D., and Wolffe, A.P. (2000). DNA methylation in health and disease. Nat Rev Genet 1, 11-19.
Rodenhiser, D., and Mann, M. (2006). Epigenetics and human disease: translating basic biology into clinical applications. CMAJ 174, 341-348.
Shah, N., and Sukumar, S. (2010). The Hox genes and their roles in oncogenesis. Nat Rev Cancer 10, 361-371.
Sham, J.S., Wei, W.I., Zong, Y.S., Choy, D., Guo, Y.Q., Luo, Y., Lin, Z.X., and Ng, M.H. (1990). Detection of subclinical nasopharyngeal carcinoma by fibreoptic endoscopy and multiple biopsy. Lancet 335, 371-374.
Slack, A., Cervoni, N., Pinard, M., and Szyf, M. (1999). DNA methyltransferase is a downstream effector of cellular transformation triggered by simian virus 40 large T antigen. J Biol Chem 274, 10105-10112.
Tcherkezian, J., and Lamarche-Vane, N. (2007). Current knowledge of the large RhoGAP family of proteins. Biol Cell 99, 67-86.
Tong, J.H., Ng, D.C., Chau, S.L., So, K.K., Leung, P.P., Lee, T.L., Lung, R.W., Chan, M.W., Chan, A.W., Lo, K.W., et al. (2010). Putative tumour-suppressor gene DAB2 is frequently down regulated by promoter hypermethylation in nasopharyngeal carcinoma. BMC Cancer 10, 253.
Tsai, C.L., Li, H.P., Lu, Y.J., Hsueh, C., Liang, Y., Chen, C.L., Tsao, S.W., Tse, K.P., Yu, J.S., and Chang, Y.S. (2006). Activation of DNA methyltransferase 1 by EBV LMP1 Involves c-Jun NH(2)-terminal kinase signaling. Cancer Res 66, 11668-11676.
Tsao, S.W., Tramoutanis, G., Dawson, C.W., Lo, A.K., and Huang, D.P. (2002a). The significance of LMP1 expression in nasopharyngeal carcinoma. Semin Cancer Biol 12, 473-487.
Tsao, S.W., Wang, X., Liu, Y., Cheung, Y.C., Feng, H., Zheng, Z., Wong, N., Yuen, P.W., Lo, A.K., Wong, Y.C., et al. (2002b). Establishment of two immortalized nasopharyngeal epithelial cell lines using SV40 large T and HPV16E6/E7 viral oncogenes. Biochim Biophys Acta 1590, 150-158.
Tsurumi, T., Fujita, M., and Kudoh, A. (2005). Latent and lytic Epstein-Barr virus replication strategies. Rev Med Virol 15, 3-15.
Virmani, A.K., Tsou, J.A., Siegmund, K.D., Shen, L.Y., Long, T.I., Laird, P.W., Gazdar, A.F., and Laird-Offringa, I.A. (2002). Hierarchical clustering of lung cancer cell lines using DNA methylation markers. Cancer Epidemiol Biomarkers Prev 11, 291-297.
Weinberg, R.A. (1994). Oncogenes and tumor suppressor genes. CA Cancer J Clin 44, 160-170.
Wittwer, C.T., Reed, G.H., Gundry, C.N., Vandersteen, J.G., and Pryor, R.J. (2003). High-resolution genotyping by amplicon melting analysis using LCGreen. Clin Chem 49, 853-860.
Wojdacz, T.K., and Dobrovic, A. (2007). Methylation-sensitive high resolution melting (MS-HRM): a new approach for sensitive and high-throughput assessment of methylation. Nucleic Acids Res 35, e41.
Yao, K.T., Zhang, H.Y., Zhu, H.C., Wang, F.X., Li, G.Y., Wen, D.S., Li, Y.P., Tsai, C.H., and Glaser, R. (1990). Establishment and characterization of two epithelial tumor cell lines (HNE-1 and HONE-1) latently infected with Epstein-Barr virus and derived from nasopharyngeal carcinomas. Int J Cancer 45, 83-89.
Yoder, J.A., Soman, N.S., Verdine, G.L., and Bestor, T.H. (1997). DNA (cytosine-5)-methyltransferases in mouse cells and tissues. Studies with a mechanism-based probe. J Mol Biol 270, 385-395.
Young, L.S., Dawson, C.W., Clark, D., Rupani, H., Busson, P., Tursz, T., Johnson, A., and Rickinson, A.B. (1988). Epstein-Barr virus gene expression in nasopharyngeal carcinoma. J Gen Virol 69 ( Pt 5), 1051-1065.
Young, L.S., and Rickinson, A.B. (2004). Epstein-Barr virus: 40 years on. Nat Rev Cancer 4, 757-768.
Yu, B., Yang, H., Zhang, C., Wu, Q., Shao, Y., Zhang, J., Guan, M., Wan, J., and Zhang, W. (2010). High-resolution melting analysis of PCDH10 methylation levels in gastric, colorectal and pancreatic cancers. Neoplasma 57, 247-252.
Yu, M.C., Ho, J.H., Ross, R.K., and Henderson, B.E. (1981). Nasopharyngeal carcinoma in Chinese---salted fish or inhaled smoke? Prev Med 10, 15-24.
Yuan, B.Z., Jefferson, A.M., Baldwin, K.T., Thorgeirsson, S.S., Popescu, N.C., and Reynolds, S.H. (2004). DLC-1 operates as a tumor suppressor gene in human non-small cell lung carcinomas. Oncogene 23, 1405-1411.
Yuan, B.Z., Miller, M.J., Keck, C.L., Zimonjic, D.B., Thorgeirsson, S.S., and Popescu, N.C. (1998). Cloning, characterization, and chromosomal localization of a gene frequently deleted in human liver cancer (DLC-1) homologous to rat RhoGAP. Cancer Res 58, 2196-2199.
Zhang, W., Li, T., Shao, Y., Zhang, C., Wu, Q., Yang, H., Zhang, J., Guan, M., Yu, B., and Wan, J. (2010). Semi-quantitative detection of GADD45-gamma methylation levels in gastric, colorectal and pancreatic cancers using methylation-sensitive high-resolution melting analysis. J Cancer Res Clin Oncol 136, 1267-1273.
Zhang, X.P.D., Liu, H.P.D., Li, B.M., Huang, P.M., Shao, J.P.D., and He, Z.P.D. (2012). Tumor suppressor BLU inhibits proliferation of nasopharyngeal carcinoma cells by regulation of cell cycle, c-Jun N-terminal kinase and the cyclin D1 promoter. BMC Cancer 12, 267.
Zhou, X., Thorgeirsson, S.S., and Popescu, N.C. (2004). Restoration of DLC-1 gene expression induces apoptosis and inhibits both cell growth and tumorigenicity in human hepatocellular carcinoma cells. Oncogene 23, 1308-1313.