臺灣博碩士論文加值系統

DNA甲基化作用在基因轉錄作用過程中扮演著重要的角色。研究發現當發生異常的甲基化作用時就會促使癌症的發生，包括口腔癌。甲基化敏感性高解析熔解分析法(MS-HRM)是一種可偵測到少量並定量DNA甲基化程度的高敏感度分析方法。本研究主要是建立MS-HRM方法來偵測臨床口腔癌組織中基因甲基化的情形。在本研究中此方法的靈敏度可偵測到1.25 ng經由亞硫酸鹽處理過後的基因體DNA。我們收集了臨床上已確診並已分期的口腔癌病人的組織檢體，藉由MS-HRM方法，分別偵測實驗室之前研究以甲基化基因晶片偵測出的一個hyper-methylated與hypo-methylated的基因，來分析臨床口腔癌組織中基因甲基化程度的情形。研究結果發現在呈現高度甲基化的RARB基因，在口腔癌組織中其甲基化程度大多高於正常組織的表現，且發現其基因甲基化的情形與腫瘤早期發展有關。另一個低度甲基化的PTHrP基因，在口腔癌組織中其甲基化程度大多低於正常組織的表現，且其甲基化程度與臨床TNM分期有關。我們發展了一個具敏感度且可定量臨床檢體DNA甲基化程度的技術。藉由不同的基因甲基化程度的偵測結果，期望能更瞭解口腔癌的致病機制，進一步協助醫師臨床上的診斷。

DNA methylation plays an important role in the process of gene transcription and regulation. It has been reported that aberrant methylation renders the occurrence of cancer, including oral cancer. Methylation-Specific PCR (MS-PCR) followed by HRM analysis (MS-HRM) provides a highly sensitive method for the detection of low level methylation and quantification of methylated DNA. The object of this study is to establish the MS-HRM method to detect the DNA methylated level in clinical oral cancer samples. The detection limitation is 1.25 ng of bisulfite genomic DNA. The clinical oral cancer tissues were collected from the patients whose progressions have been well diagnosed and staged. One hyper-methylated and one hypo-methylated candidate gene which previously identified by CpG island microarray were selected to detect the methylation level in clinical oral cancer tissues by MS-HRM method. For hyper-methylated gene, RARB, the frequency of the gene methylation in oral cancer tissue was significantly higher than that in normal tissues. Moreover, the gene methylation status is associated with early step of tumor progression. On the other hand, the hypo-methylated gene, PTHrP, the frequency of the gene methylation in oral cancer tissue was significantly lower than that in normal tissues. Moreover, the gene methylation status is associated with tumor-node-metastasis staging in oral cancer samples. Taken together, we develop a sensitive and quantitative method to detect DNA methylation level in clinical sample. Our results may shed a light to understand the pathogenesis of oral cancer and assistant advanced in clinical diagnosis for oral cancer.

誌謝 i
中文摘要 iii
英文摘要 iv
目錄 v
表目錄 viii
圖目錄 ix
第一章 前言 1
第一節 研究背景 1
1.1 口腔癌 1
1.1.1 臨床症狀與流行病學 1
1.1.2 臨床與病理分期 2
1.1.3 診斷與預後 3
1.2 基因甲基化修飾機制及對癌症細胞的影響 3
1.2.1 基因甲基化修飾機制 3
1.2.2 基因甲基化對癌症細胞的影響 5
1.2.3 檢測基因甲基化之技術發展 6
1.2.4 基因甲基化在口腔癌動物模式的表現 8
1.2.4.1 基因甲基化在口腔癌動物模式的表現 8
1.2.4.2 細胞週期蛋白依賴性激酶抑制因子2A基因 (cyclin-dependent kinase inhibitor 2A；CDKN2A；p16) 9
1.2.4.3 視網酸受體基因(beta-retinoic acid receptor ; RARB) 10
1.2.4.4 副甲狀腺素相關蛋白質(parathyroid-Related Protein；PTHrP) 11
第二節 研究目的 13
第二章 研究方法 14
第一節 研究設計 14
第二節 研究材料 14
2.2.1 檢體 14
2.2.2 引子(primer) 14
第三節 實驗方法 15
2.3.1 DNA萃取 15
2.3.2 全基因組擴增法(whole-genome amplification；WGA) 15
2.3.3 亞硫酸鹽處理(bisulfite modification) 16
2.3.4 甲基化敏感高解析熔解分析法(methylation-sensitive high- resolution Melting Analysis；MS-HRM) 17
2.3.5 電泳分析(gel electrophoresis analysis) 17
2.3.6 亞硫酸鹽定序(Bisulfite sequencing) 17
2.3.7 統計分析 18

第三章 研究結果 19
3.1 基因體DNA 全甲基化及未甲基化之Melting標準曲線 19
3.2 MS-HRM 可偵測到的limitation 20
3.3 p16基因可以MS-HRM進行甲基化定量偵測 20
3.4 RARB基因可以MS-HRM進行甲基化定量偵測 20
3.5 PTHrP基因可以MS-HRM進行甲基化定量偵測 21
3.6 統計分析臨床口腔癌組織中RARB基因甲基化的表現 22
3.7 統計分析臨床口腔癌組織中RARB基因甲基化表現與TNM分期的關係 22
3.8 統計分析臨床口腔癌組織中PTHrP基因甲基化的表現 23
3.9 統計分析臨床口腔癌組織中PTHrP基因甲基化表現與TNM分期的關係 23
第四章 討論 25
第五章 結論與建議 29
第一節 結論 29
第二節 建議 29
第六章 參考文獻 30
附錄 48

表目錄
表 一：基因引子的序列、PCR黏合溫度、片段大小及總CpG數 34
表 二：以RARB基因進行內部品管與外部品管分析結果 35
表 三：以PTHrP基因進行內部品管與外部品管分析結果 36

圖目錄
圖 一：基因體DNA全甲基化及未甲基化的定序結果 38
圖 二：基因體DNA全甲基化及未甲基化之Melting參考標準 39
圖 三：MS-HRM 可偵測到的limitation結果 40
圖 四：p16基因可以MS-HRM進行甲基化定量偵測 41
圖 五：RARB基因可以MS-HRM進行甲基化定量偵測 42
圖 六：PTHrP 基因可偵測到的Quantitative Sensitivity結果及再現性 43
圖 七：統計分析臨床口腔癌組織中RARB基因甲基化的表現 44
圖 八：統計分析臨床口腔癌組織中RARB基因甲基化表現與TNM分期的關係 45
圖 九：統計分析臨床口腔癌組織中PTHrP基因甲基化的表現 46
圖 十：統計分析臨床口腔癌組織中PTHrP基因甲基化表現與TNM分期的關係 47

1.Agrawal, A., Murphy, R.F., and Agrawal, D.K. (2007). DNA methylation in breast and colorectal cancers. Mod Pathol 20, 711-721.
2.Baba, S., Hara, A., Kato, K., Long, N.K., Hatano, Y., Kimura, M., Okano, Y., Yamada, Y., and Shibata, T. (2009). Aberrant promoter hypermethylation of the CHFR gene in oral squamous cell carcinomas. Oncol Rep 22, 1173-1179.
3.Braggio, E., Maiolino, A., Gouveia, M.E., Magalhaes, R., Souto Filho, J.T., Garnica, M., Nucci, M., and Renault, I.Z. (2010). Methylation status of nine tumor suppressor genes in multiple myeloma. Int J Hematol 91, 87-96.
4.Carter, L.M., Harris, A.T., Kavi, V.P., Johnson, S., and Kanatas, A. (2009). Oral cancer awareness amongst hospital nursing staff: a pilot study. BMC Oral Health 9, 4.
5.Chakravarti, N., Mathur, M., Bahadur, S., Shukla, N.K., Rochette-Egly, C., and Ralhan, R. (2001). Expression of RARalpha and RARbeta in human oral potentially malignant and neoplastic lesions. Int J Cancer 91, 27-31.
6.Chen, K., Sawhney, R., Khan, M., Benninger, M.S., Hou, Z., Sethi, S., Stephen, J.K., and Worsham, M.J. (2007). Methylation of multiple genes as diagnostic and therapeutic markers in primary head and neck squamous cell carcinoma. Arch Otolaryngol Head Neck Surg 133, 1131-1138.
7.Csepregi, A., Ebert, M.P., Rocken, C., Schneider-Stock, R., Hoffmann, J., Schulz, H.U., Roessner, A., and Malfertheiner, P. (2010). Promoter methylation of CDKN2A and lack of p16 expression characterize patients with hepatocellular carcinoma. BMC Cancer 10, 317.
8.Dunn, B.K. (2003). Hypomethylation: one side of a larger picture. Ann N Y Acad Sci 983, 28-42.
9.Eads, C.A., Danenberg, K.D., Kawakami, K., Saltz, L.B., Blake, C., Shibata, D., Danenberg, P.V., and Laird, P.W. (2000). MethyLight: a high-throughput assay to measure DNA methylation. Nucleic Acids Res 28, E32.
10.Ganderton, R.H., and Briggs, R.S. (1997). CpG island methylation and promoter usage in the parathyroid hormone-related protein gene of cultured lung cells. Biochim Biophys Acta 1352, 303-310.
11.Ganderton, R.H., and Briggs, R.S. (2000). Increased upstream methylation has no influence on the overexpression of the parathyroid hormone-related protein gene in squamous cell carcinoma of the lung. European journal of cancer (Oxford, England : 1990) 36, 2128-2136.
12.Guo, X.L., Sun, S.Z., Wang, W.X., Wei, F.C., Yu, H.B., and Ma, B.L. (2007). Alterations of p16INK4a tumour suppressor gene in mucoepidermoid carcinoma of the salivary glands. Int J Oral Maxillofac Surg 36, 350-353.
13.Hayashi, K., Yokozaki, H., Naka, K., Yasui, W., Lotan, R., and Tahara, E. (2001). Overexpression of retinoic acid receptor beta induces growth arrest and apoptosis in oral cancer cell lines. Jpn J Cancer Res 92, 42-50.
14.Herman, J.G., Graff, J.R., Myohanen, S., Nelkin, B.D., and Baylin, S.B. (1996). Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci U S A 93, 9821-9826.
15.Huang, S.H., Lee, H.S., Mar, K., Ji, D.D., Huang, M.S., and Hsia, K.T. (2010). Loss expression of O6-methylguanine DNA methyltransferase by promoter hypermethylation and its relationship to betel quid chewing in oral squamous cell carcinoma. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 109, 883-889.
16.Irvin D. Fleming, J.S.C., Donald Earl Henson, Robert V. P. Hutter, B. J. Kennedy, Gerald P. Murphy, Brian O’Sulivan, Leslie H. Sobin, John W. Yarbro. (1997). AJCC cancer stage manual., Fifth Edition edn.
17.Kordi-Tamandani, D.M., Moazeni-Roodi, A., Rigi Ladez, M.A., Hashemi, M., Birjandian, E., and Torkamanzehi, A. (2010). Analysis of methylation patterns and expression profiles of p14ARF gene in patients with oral squamous cell carcinoma. Int J Biol Markers 25, 99-103.
18.Kristensen, L.S., Mikeska, T., Krypuy, M., and Dobrovic, A. (2008). Sensitive Melting Analysis after Real Time- Methylation Specific PCR (SMART-MSP): high-throughput and probe-free quantitative DNA methylation detection. Nucleic Acids Res 36, e42.
19.Lee, C.H., Ko, Y.C., Huang, H.L., Chao, Y.Y., Tsai, C.C., Shieh, T.Y., and Lin, L.M. (2003). The precancer risk of betel quid chewing, tobacco use and alcohol consumption in oral leukoplakia and oral submucous fibrosis in southern Taiwan. Br J Cancer 88, 366-372.
20.Liu, W., Guan, M., Su, B., Li, J., Ma, W., Liu, C., Li, M., Lin, Y., and Lu, Y. (2010). Rapid determination of AKAP12 promoter methylation levels in peripheral blood using methylation-sensitive high resolution melting (MS-HRM) analysis: application in colorectal cancer. Clinica chimica acta; international journal of clinical chemistry 411, 940-946.
21.Malentacchi, F., Forni, G., Vinci, S., and Orlando, C. (2009). Quantitative evaluation of DNA methylation by optimization of a differential-high resolution melt analysis protocol. Nucleic Acids Res 37, e86.
22.Moseley, J.M., and Gillespie, M.T. (1995). Parathyroid hormone-related protein. Crit Rev Clin Lab Sci 32, 299-343.
23.Nakahara, Y., Shintani, S., Mihara, M., Hino, S., and Hamakawa, H. (2006). Detection of p16 promoter methylation in the serum of oral cancer patients. Int J Oral Maxillofac Surg 35, 362-365.
24.Narayan, G., Arias-Pulido, H., Koul, S., Vargas, H., Zhang, F.F., Villella, J., Schneider, A., Terry, M.B., Mansukhani, M., and Murty, V.V. (2003). Frequent promoter methylation of CDH1, DAPK, RARB, and HIC1 genes in carcinoma of cervix uteri: its relationship to clinical outcome. Mol Cancer 2, 24.
25.Ohta, S., Uemura, H., Matsui, Y., Ishiguro, H., Fujinami, K., Kondo, K., Miyamoto, H., Yazawa, T., Danenberg, K., Danenberg, P.V., et al. (2009). Alterations of p16 and p14ARF genes and their 9p21 locus in oral squamous cell carcinoma. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 107, 81-91.
26.Paulsson, K., An, Q., Moorman, A.V., Parker, H., Molloy, G., Davies, T., Griffiths, M., Ross, F.M., Irving, J., Harrison, C.J., et al. (2009). Methylation of tumour suppressor gene promoters in the presence and absence of transcriptional silencing in high hyperdiploid acute lymphoblastic leukaemia. Br J Haematol 144, 838-847.
27.Rand, K., Qu, W., Ho, T., Clark, S.J., and Molloy, P. (2002). Conversion-specific detection of DNA methylation using real-time polymerase chain reaction (ConLight-MSP) to avoid false positives. Methods 27, 114-120.
28.Shaw, R.J., Liloglou, T., Rogers, S.N., Brown, J.S., Vaughan, E.D., Lowe, D., Field, J.K., and Risk, J.M. (2006). Promoter methylation of P16, RARbeta, E-cadherin, cyclin A1 and cytoglobin in oral cancer: quantitative evaluation using pyrosequencing. Br J Cancer 94, 561-568.
29.Silva, M., Azenha, D., Pereira, C., Almeida, A., Balseiro, S., Sampaio, A.M., Santos, P., and Carvalho, L. (2010). [Gastric carcinoma and chronic gastritis: epigenetic regulation of CDH1 (E-Cadherin), CDKN2A (p16INK4A), PTGS2 (COX-2) and EGFR genes through methylation]. Acta Med Port 23, 5-14.
30.Singal, R., and Ginder, G.D. (1999). DNA methylation. Blood 93, 4059-4070.
31.Smith, E., Jones, M.E., and Drew, P.A. (2009). Quantitation of DNA methylation by melt curve analysis. BMC Cancer 9, 123.
32.Southby, J., Kissin, M.W., Danks, J.A., Hayman, J.A., Moseley, J.M., Henderson, M.A., Bennett, R.C., and Martin, T.J. (1990). Immunohistochemical localization of parathyroid hormone-related protein in human breast cancer. Cancer Res 50, 7710-7716.
33.Steinmann, K., Sandner, A., Schagdarsurengin, U., and Dammann, R.H. (2009). Frequent promoter hypermethylation of tumor-related genes in head and neck squamous cell carcinoma. Oncol Rep 22, 1519-1526.
34.Sun, S.Y. (2004). Retinoic acid receptor beta and colon cancer. Cancer Biol Ther 3, 87-88.
35.Takayama, Y., Mori, T., Nomura, T., Shibahara, T., and Sakamoto, M. (2010). Parathyroid-related protein plays a critical role in bone invasion by oral squamous cell carcinoma. Int J Oncol 36, 1387-1394.
36.Warnecke, P.M., Stirzaker, C., Song, J., Grunau, C., Melki, J.R., and Clark, S.J. (2002). Identification and resolution of artifacts in bisulfite sequencing. Methods 27, 101-107.
37.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.
38.Xiong, Z., and Laird, P.W. (1997). COBRA: a sensitive and quantitative DNA methylation assay. Nucleic Acids Res 25, 2532-2534.
39.Yakushiji, T., Noma, H., Shibahara, T., Arai, K., Yamamoto, N., Tanaka, C., Uzawa, K., and Tanzawa, H. (2001). Analysis of a role for p16/CDKN2 expression and methylation patterns in human oral squamous cell carcinoma. Bull Tokyo Dent Coll 42, 159-168.