臺灣博碩士論文加值系統

鼻竇倒生性乳突瘤具有易復發、侵犯周遭組織以及和扁平細胞癌相關連等特性。本研究旨在利用微陣列晶片研究基因表現差異、分析DLEC1在倒生性乳突瘤腫瘤發生和癌化的角色，以及探討人類乳突病毒在倒生性乳突瘤和扁平細胞癌中是否和DLEC1表現抑制相關。
在本前瞻性研究中，我們在三組檢體 – 扁平細胞癌 (n=5)、倒生性乳突瘤(n=5)、正常鼻粘膜 (n=5) – 利用微陣列晶片及後續之即時定量聚合酶連鎖反應來分析DLEC1轉錄物之基因表現差異。為了進一步探討其機轉，我們採用甲基化特異聚合酶連鎖反應及後續之直接定序方法，研究這三組檢體中DLEC1啟動子之甲基化程度。另外我們利用免疫化學方法進行DLEC1表現的驗證 (三組檢體皆n=25)。人類乳突病毒的感染及亞型採用巢式聚合酶連鎖反應來檢測。
在結果方面，和正常鼻粘膜比較，DLEC1在倒生性乳突瘤及扁平細胞癌皆向下調控 (在即時定量聚合酶連鎖反應和免疫化學方法皆p<0.01)，在扁平細胞癌的表現又比倒生性乳突瘤低下 (即時定量聚合酶連鎖反應: p<0.01、免疫化學方法：p=0.024)。DLEC1在扁平細胞癌的表現抑制是經由啟動子過度甲基化機轉，但在倒生性乳突瘤中無此現象。在兩組腫瘤檢體中，人類乳突病毒的感染與否並沒有影響DLEC1的表現。
總結來說，在鼻竇扁平細胞癌及倒生性乳突瘤中，DLEC1表現皆向下調控但機轉不同，DLEC1在扁平細胞癌的表現抑制是經由啟動子過度甲基化，但在倒生性乳突瘤無此機轉。人類乳突病毒於兩組腫瘤中DLEC1的表現抑制並無角色。

Sinonasal inverted papilloma (IP) is known for a tendency to recur, invasion of surrounding tissues, and an association with squamous cell carcinoma (SCC). This study aimed to investigate differential gene expression profile using microarrays, dissect the role of the DLEC1 in the tumorigenesis and carcinogenesis of IP, and analyze if human papilloma virus (HPV) in the IP and SCC samples was associated with repression of DLEC1.
In our prospective study, we analyzed differentially expressed transcripts of DLEC1 in SCC (n=5), IP (n=5) and normal mucosa (n=5) using microarrays and further validating by quantitative real-time polymerase(QRT-PCR) chain reaction. To further examine the mechanism, methylation-specific polymerase chain reactions and subsequent autosequencing were also used to examine the methylation status of DLEC1 promoter in the same samples as the microarray training set. To examine the expression of DLEC1 in a large validation set, it was evaluated by immunohistochemistry (n=25 in SCC, IP and normal mucosa). HPV subtypes were determined by nested PCR.
DLEC1 was down-regulated in IP and SCC tissues compared to normal mucosa (p < 0.01 both in immunohistochemistry and QRT-PCR), with SCC more repressed (p < 0.01, p = 0.024, respectively in QRT-PCR and immunohistochemistry) than IP tissues. Repression of DLEC1 in SCC tissues was via promoter hypermethylation, which was not observed in IP tissues. No significant difference of DLEC1 expression was observed in HPV-positive and HPV-negative IP and SCC tissues.
In conclusion, In sinonasal SCC and IP, DLEC1 was down-regulated but exhibited a distinct mechanism. Inactivation of DLEC1 in SCC occurred through DNA methylation, whereas this mechanism was not observed in IP. HPV infection was not observed to have a role in the repression of DLEC1.

CHAPTER I Introduction…………………………………………… 1
1.1 Clinicopathologic studies in sinonasal inverted papilloma……………………………………………………1
1.2 HPV and inverted papilloma……………………… 2
1.2.1 The role of HPV in the pathogenesis of IP………………2
1.2.2 The role of HPV in the carcinogenesis of IP……2
1.3 Molecular biologic study of IP and related SCC………3
1.4 Microarray studies in sinonasal tissues……………………4
1.5 A tumor suppressor gene- DLEC1 ……………………………5
1.6 Aims…………………………………………………………7
CHAPTER II Experimental design.................................... 8
2.1 Study design and patients………………................. 8
2.2 RNA extraction, microarray data processing, Affymetrix gene chip analysis and Affymetrix genome-wide human SNP Nsp/Sty assay kit 6.0 analysis................................................ 8
2.3 QRT-PCR………………………………………………………… 9
2.4 MS-PCR………………………………………………… 10
2.5 TOPO cloning…………………………………………………11
2.6 Autosequencing………………………………………… 11
2.7 Immunohistochemistry (IHC)………………………………….11
2.7.1 Generation of DLEC1 polyclonal antibody………11
2.7.2 IHC protocol……………………………………………12
2.7.3 Semi-quantification of immuno-staining………………13
2.8 HPV typing……………………………………………………..13
2.8.1 HPV genotyping with genechip…………………………13
2.8.2 DNA extraction…………………………………………14
2.8.3 Nested PCR………………………………………………14
2.9 Statistical analysis……………………………………………15
CHAPTER III Results............................................ 16
3.1 Transcripts of DLEC1 in SCC, IP and normal tissues evaluated by Affymetrix U133A genechip and RTQ-PCR………16
3.2 DLEC1 is significantly repressed in IP and SCC as determined using IHC…………………………………………………17
3.3 Promoter methylation in DLEC1 in sinonasal SCC…………18
3.4 The relationship of HPV involvement with DLEC1 repression in IP and SCC………………………………………………19
CHAPTER IV Discussion…………………………………………20
4.1 Microarray analysis of sinonasal IP, SCC and normal mucosa..20
4.2 The role of p53 in the carcinongenesis of IP……………20
4.3 Epigenetic modulation in sinonasal IP and SCC…………21
4.4 Researches of DLEC1 methylation in malignant tumors…21
4.5 DLEC1 involvement in the pathogenesis of IP and SCC…22
4.6 The relationship between HPV and DLEC1 in IP and SCC…23
4.7 The different traits of HPV in head and neck cancer and cervical cancer………………………………………………………23
CHAPTER V Conclusion............................................ 25
References............................................ 26
Tables............................................... 31
Table 1 Articles regarding to the role and regulatory mechanism of DLEC1 in various tumors………………………………31
Table 2 Primers and amplicon size of MY 9/11 and GP5+/6….. 33
Table 3 Demographic data of the patients with samples processed with cDNA microarray……………………………………34
Table 4 The most differentially expressed genes in sinonasal inverted papilloma and squamous cell carcinoma…36
Table 5 Demographic data of the patients receiving immunohistochemistry study……………………………40
Table 6 Scores and positive rates of immunohistochemistry…………………………41
Table 7 Detection of HPV subtypes in IP and SCC tissue samples with Easychip® HPV Blot and nested PCR individually…42
Table 8 Threshold cycle difference (CT) of quantitative real-time PCR of IP and SCC samples with or without HPV infection…43
Figures………………………………………………………………44
Fig. 1. The histopathological character of inverted papillomas……44
Fig. 2. Organization of HPV genome……………………………45
Fig. 3. Proposed molecular model of carcinogenesis in head and neck SCC……………………………………………………46
Fig. 4. Layout of HPV genome……………………………… 47
Fig. 5. RNA quality test in this study…………………… 48
Fig. 6. The agilent test of this study……………………49
Fig. 7. Principal Component Analysis analyzed from microarray result………………………………………………… 50
Fig. 8.Differential expression profiles in normal, IP and SCC analyzed by hierarchical clustering……………………………………51
Fig. 9. SNP Nsp/Sty assay kit 6.0 analysis result related to DLEC1 areas……………………………………………52
Fig. 10. Transcripts of DLEC1 analyzed by QRT-PCR…………53
Fig. 11. DLEC1 expression level detected by IHC…………54
Fig. 12. Methylated status of DLEC1 promoter and exon1 by bisulfite-PCR…………………………………………55
Fig. 13. Methylation status of DLEC1 promoter and exon 1 after direct sequencing………………………………………56
Fig. 14. The result of Easychip HPV blot on which subtypes of HPV are shown………………………………………………57
Fig. 15. MetaCore analysis of transcriptome in IP samples…………58
Fig. 16. Mutations in p53 in our SCC samples…………59

1. Sauter A, Matharu R, Hormann K, Naim R. Current advances in the basic research and clinical management of sinonasal inverted papilloma (review). Oncol Rep 2007;17(3):495-504.
2. Buchwald C, Franzmann MB, Tos M. Sinonasal papillomas: a report of 82 cases in Copenhagen County, including a longitudinal epidemiological and clinical study. Laryngoscope 1995;105(1):72-9.
3. Osguthorpe JD, Richardson M. Frontal sinus malignancies. Otolaryngol Clin North Am 2001;34(1):269-81.
4. Tang AC, Grignon DJ, MacRae DL. The association of human papillomavirus with Schneiderian papillomas: a DNA in situ hybridization study. J Otolaryngol 1994;23(4):292-7.
5. Syrjanen KJ. HPV infections in benign and malignant sinonasal lesions. J Clin Pathol 2003;56(3):174-81.
6. Lawson W, Schlecht NF, Brandwein-Gensler M. The role of the human papillomavirus in the pathogenesis of Schneiderian inverted papillomas: an analytic overview of the evidence. Head Neck Pathol 2008;2(2):49-59.
7. McKay SP, Gregoire L, Lonardo F, Reidy P, Mathog RH, Lancaster WD. Human papillomavirus (HPV) transcripts in malignant inverted papilloma are from integrated HPV DNA. Laryngoscope 2005;115(8):1428-31.
8. Al Sarakbi W, Reefy S, Jiang WG, Roberts T, Newbold RF, Mokbel K. Evidence of a tumour suppressor function for DLEC1 in human breast cancer. Anticancer Res 2010;30(4):1079-82.
9. Schwerer MJ, Sailer A, Kraft K, Baczako K, Maier H. Differentiation-related p53 protein expression in nondysplastic sinonasal inverted papillomas. Am J Rhinol 2001;15(5):347-51.
10. Hardisson D. Molecular pathogenesis of head and neck squamous cell carcinoma. Eur Arch Otorhinolaryngol 2003;260(9):502-8.
11. Califano J, van der Riet P, Westra W, Nawroz H, Clayman G, Piantadosi S, et al. Genetic progression model for head and neck cancer: implications for field cancerization. Cancer Res 1996;56(11):2488-92.
12. Califano J, Koch W, Sidransky D, Westra WH. Inverted sinonasal papilloma : a molecular genetic appraisal of its putative status as a Precursor to squamous cell carcinoma. Am J Pathol 2000;156(1):333-7.
13. Huang CC, Lee TJ, Chang PH, Lee YS, Chuang CC, Jhang YJ, et al. Desmoglein 3 is overexpressed in inverted papilloma and squamous cell carcinoma of sinonasal cavity Laryngoscope 2010;120(1):26-9.
14. Chao JC, Fang SY. Expression of epidermal growth factor receptor in the inverted papilloma and squamous cell carcinoma of nasal cavity. Eur Arch Otorhinolaryngol 2008;265(8):917-22.
15. Koo BS, Jung BJ, Kim SG, Liang ZL, Yeong MK, Rha KS. Altered expression of E-cadherin and β-catenin in malignant transformation of sinonasal inverted papillomas. Rhinology 2011;49(4):479-85.
16. Katori H, Nozawa A, Tsukuda M. Markers of malignant transformation of sinonasal inverted papilloma. Eur J Surg Oncol 2005;31(8):905-11.
17. Liu Z, Niu Y, Li C, Yang Y, Gao C. Integrating multiple microarray datasets on oral squamous cell carcinoma to reveal dysregulated networks. Head Neck 2011 Dec Epub.
18. Liu Z, Kim J, Sypek JP, Wang IM, Horton H, Oppenheim FG, et al. Gene expression profiles in human nasal polyp tissues studied by means of DNA microarray. J Allergy Clin Immunol 2004;114(4):783-90.
19. Choi P, Chen C. Genetic expression profiles and biologic pathway alterations in head and neck squamous cell carcinoma. Cancer 2005;104(6):1113-28.
20. Chan WH, Chang KP, Yang SW, Yao TC, Ko TY, Lee YS, et al. Transcriptional repression of DLEC1 associates with the depth of tumor invasion in oral squamous cell carcinoma. Oral Oncol 2010;46(12):874-9.
21. Kwong J, Chow LS, Wong AY, Hung WK, Chung GT, To KF, et al. Epigenetic inactivation of the deleted in lung and esophageal cancer 1 gene in nasopharyngeal carcinoma. Genes Chromosomes Cancer 2007;46(2):171-80.
22. Kwong J, Lee JY, Wong KK, Zhou X, Wong DT, Lo KW, et al. Candidate tumor-suppressor gene DLEC1 is frequently downregulated by promoter hypermethylation and histone hypoacetylation in human epithelial ovarian cancer. Neoplasia 2006;8(4):268-78.
23. Qiu GH, Salto-Tellez M, Ross JA, Yeo W, Cui Y, Wheelhouse N, et al. The tumor suppressor gene DLEC1 is frequently silenced by DNA methylation in hepatocellular carcinoma and induces G1 arrest in cell cycle. J Hepatol 2008;48(3):433-41.
24. Seng TJ, Currey N, Cooper WA, Lee CS, Chan C, Horvath L, et al. DLEC1 and MLH1 promoter methylation are associated with poor prognosis in non-small cell lung carcinoma. Br J Cancer 2008;99(2):375-82.
25. Ying J, Poon FF, Yu J, Geng H, Wong AH, Qiu GH, et al. DLEC1 is a functional 3p22.3 tumour suppressor silenced by promoter CpG methylation in colon and gastric cancers. Br J Cancer 2009;100(4):663-9.
26. Smith IM, Mithani SK, Liu C, Chang SS, Begum S, Dhara M, et al. Novel integrative methods for gene discovery associated with head and neck squamous cell carcinoma development. Arch Otolaryngol Head Neck Surg 2009;135(5):487-95.
27. Fukuoka J, Dracheva T, Shih JH, Hewitt SM, Fujii T, Kishor A, et al. Desmoglein 3 as a prognostic factor in lung cancer. Hum Pathol 2007;38(2):276-83.
28. Lai CH, Huang HJ, Hsueh S, Chao A, Lin CT, Huang SL, et al. Human papillomavirus genotype in cervical cancer: a population-based study. Int J Cancer 2007;120(9):1999-2006.
29. Huang HJ, Huang SL, Lin CY, Lin RW, Chao FY, Chen MY, et al. Human papillomavirus genotyping by a polymerase chain reaction-based genechip method in cervical carcinoma treated with neoadjuvant chemotherapy plus radical surgery. Int J Gynecol Cancer 2004;14(4):639-49.
30. Poeta ML, Manola J, Goldwasser MA, Forastiere A, Benoit N, Califano JA, et al. TP53 mutations and survival in squamous-cell carcinoma of the head and neck. N Engl J Med 2007;357(25):2552-61.
31. Chen K, Sawhney R, Khan M, Benninger MS, Hou Z, Sethi S, et al. Methylation of multiple genes as diagnostic and therapeutic markers in primary head and neck squamous cell carcinoma. Arch Otolaryngol Head Neck Surg 2007;133(11):1131-8.
32. Stephen JK, Vaught LE, Chen KM, Sethi S, Shah V, Benninger MS, et al. Epigenetic events underlie the pathogenesis of sinonasal papillomas. Mod Pathol 2007;20(10):1019-27.
33. Daigo Y, Nishiwaki T, Kawasoe T, Tamari M, Tsuchiya E, Nakamura Y. Molecular cloning of a candidate tumor suppressor gene, DLC1, from chromosome 3p21.3. Cancer Res 1999;59(8):1966-72.
34. Katori H, Nozawa A, Tsukuda M. Increased expression of matrix metalloproteinase-2 and 9 and human papilloma virus infection are associated with malignant transformation of sinonasal inverted papilloma. J Surg Oncol 2006;93(1):80-5.
35. Munoz N, Bosch FX, de Sanjose S, Herrero R, Castellsague X, Shah KV, et al. Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 2003;348(6):518-27.
36. Gillison ML, Koch WM, Capone RB, Spafford M, Westra WH, Wu L, et al. Evidence for a causal association between human papillomavirus and a subset of head and neck cancers. J Natl Cancer Inst 2000;92(9):709-20.
37. D'Souza G, Kreimer AR, Viscidi R, Pawlita M, Fakhry C, Koch WM, et al. Case-control study of human papillomavirus and oropharyngeal cancer. N Engl J Med 2007;356(19):1944-56.
38. Zhang Y, Miao Y, Yi J, Wang R, Chen L. Frequent epigenetic inactivation of deleted in lung and esophageal cancer 1 gene by promoter methylation in non-small-cell lung cancer. Clin Lung Cancer 2010;11(4):264-70.
39. Zhang Q, Ying J, Li J, Fan Y, Poon FF, Ng KM, et al. Aberrant promoter methylation of DLEC1, a critical 3p22 tumor suppressor for renal cell carcinoma, is associated with more advanced tumor stage. J Urol 2010;184(2):731-7.
40. Zhang Y, Ye X, Geng J, Chen L. Epigenetic inactivation of deleted in lung and esophageal cancer 1 gene by promoter methylation in gastric and colorectal adenocarcinoma. Hepatogastroenterology 2010;57(104):1614-9.