Molecular mechanisms underlying the pathogenesis of nasal polyposis and its response to steroid treatment LI CHUNWEI (Bachelor of Medicine, Sun Yat-sen University, P.R China) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF OTOLARYNGOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2009 i Acknowledgement As time goes by, I have spent about years to pursue my PhD in National University of Singapore During my PhD study, I should sincerely thank my supervisor, Assoc Prof Wang De Yun He not only guides my study, but also sets an excellent example of what a good scientist should be I feel honored to be his student I must specially thank Assoc Prof Loh Kwok Seng, our head of department, who gives me a big help and support in my study and work I appreciate that he gives me the opportunity to work in our department I am grateful to Dr Cheung Wai from UCSD (San Diego) for his excellent support in my papers His scientific view and advice ultimately contributes to the publication of my papers and the improvement of my work When I joined the ENT lab in 2003, I was new to most of the lab work I need to thank my seniors, Dr Hao Jing, Dr Liang Xiao Hui, and Mr Foong Kook Heng With their selfless help in my experiment, I am familiar to many of basic research works Throughout my PhD study, we have many collaboration works The superantigen study was collaborated with Dr Mark Taylor from Department of Microbiology The methylation study was collaborated with Dr Tao Qian from Johns Hopkins Singapore I thank Dr Fu Li, Dr Liu Ding Xie, and Dr Qiu Guo Hua from Dr Tao’s lab for teaching me the methylation experiment The gene expression study was collaborated with Prof Li Tian Ying from Sun Yat-Sen University (Guangzhou, China) I especially thank Dr Lin Zhi Bin and Dr Chen Yan Qiu from Prof Li’s department for their collection and procession of the clinical samples In addition, I also thank Dr Pang Yoke-Teen from our department for providing us clinical biopsies Without their generous help and support, I could not fulfill my PhD work Other peoples, who are not our major collaborators, also have given me a great help in my work for these years Mr Lim Joe Thuan from Department of Pathology always helped me out in the histopathological experiment Ms Wen Hong Mei from ii Department of Pediatrics often did me a favor for the paraffin sectioning work Dr Yang Yan from Sun Yat-Sen University assisted me in doing experiment during the time in Guangzhou Dr Shanthi Wasser from Department of Medicine showed her generosity and let me the real-time PCR work in her lab Dr Shang Hui Sheng, Dr Ong Tan Ching, and Ms Jiang Nan from Department of Biological Science under Asst Prof Chew Fook Tim’s research group, always lent me a hand with my routine lab work My lab neighbors, Dr Huang Chiung Hui, Dr Kuo I-Chun, and Dr Seow See Voon under Prof Chua Kaw Yan’s research group, always give me assistance and let me share the facilities with them Dr Chan Yiong Huak always gives me the expert advice and help in my statistic work I should like to express my gratitude for your kindness I would like to thank my friends and colleagues from Faculty of Medicine, Zhong Fei, Li Xiujin, Li Guang, Li Yang, Ding Ying, Yang Fei, Zhang Gang, Song Guanghui, Li Xinhua, Chen Jie, Meng Qingying, Liu Qiang, Wu Qinghui, Li Chunmei, Li Chengda, Seow Weijie, Jennifer, Jason, Zhu Ganghua, Tan Shihui, Serene, Judy, Emily, and Cindy Although I have not contacted some of them for a long time, I treasure your support and friendship in my life I owe a big thank to Prof James Smith (from Oregon Health & Science University) and Ms Ho Wei Ling (from World Scientific Publishing Co.), for their generosity and help in the revision of my thesis I also thank National University of Singapore for giving me the chance to pursue PhD and offering me the scholarship And last but not least, this dissertation is dedicated to my family My parents are always standing with me and giving me endless love and encouragement My wife is always showing her patience and thoughtfulness to my work, and sharing the joy and happiness with me I feel fortunate and happy I am your son/husband Yours, Li Chunwei March, 2009, in Singapore iii Publications Li CW, Cheung W, Lin ZB, Li TY, Lim JT, Wang DY Oral steroids enhance epithelial repair in nasal polyposis via up-regulation of AP-1 gene network Thorax 2009 Jan 21 [Epub ahead of print] (Impaction Factor: 7.06) Liang XH, Cheung W, Heng CK, Liu JJ, Li CW, Lim B, Wang de Y CD14 promoter polymorphisms have no functional significance and are not associated with atopic phenotypes Pharmacogenet Genomics 2006 Apr;16(4):229-36 (Impaction Factor: 4.41) Wang DY, Li CW Control of nasal obstruction in patients with persistent allergic rhinitis Zhonghua Er Bi Yan Hou Tou Jing Wai Ke Za Zhi 2006 Sep;41(9):716-20 Li CW, Cheung W, Li TY, Lin ZB, Lim JT, Wang DY Expression profile of eosinophil- and neutrophil-associated genes in patients with nasal polyposis Article submitted Li CW, Cheung W, Pang YT, Wang DY Low level methylation of some tumor suppressor genes in nasal polyps and normal nasal mucosa Article in preparation Presentations at conferences Li CW, Pang YT, Tao Q, Wang DY Promoter methylation status of multiple genes in nasal polyps Poster presentation in 2005 World Allergy Congress, Munich, Germany Poster No 751 Li CW, Cheung W, Lin ZB, Li TY, Lim JT, Wang DY Glucocorticoids promote epithelial repair in nasal polyps via upregulating Activator protein-1 Oral presentation in XXVII Congress of the European Academy of Allergology and Clinical Immunology, EAACI 2008, Barcelona, Spain Abstract No 119 (Awarded with the best oral presentation in the session of “Inflammatory Mechanisms in Rhinosinusal Disease”.) iv Molecular mechanisms underlying the pathogenesis of nasal polyposis and its response to steroid treatment Content of the Thesis Title Page ………………………………………………………… i Acknowledgment …………………………………………………… ii Publications and Presentations at Conferences iv Table of Contents …………………………………………………… v Summary …………………………………………………………… x List of Tables ………………………………………………………… xii List of Figures ……………………………………………………… xiii List of Abbreviations ……………………………………………… xv Chapter Nasal Polyposis – a Multifactorial Chronic Inflammatory Disease (Literature review) 1.1 Histopathology ……………………………………………………………… 1.2 Epidemiology ……………………………………………………………… 1.3 Anatomy …………………………………………………………………… 1.4 Pathogenesis ………………………………………………………………… 1.4.1 Environmental factors ……………………………………………….… 1.4.2 Genetic predisposition ………………………………………………… 1.4.3 Allergy …………………………………………………………….…… v 1.4.4 Microorganisms ………………………………………………………… 1.4.5 Cellular components …………………………………………………… 11 1.4.6 Molecular chemical mediators ………………………………………… 15 1.4.7 Deregulation of fluid and electrolyte transport ………………………… 23 1.4.8 Epithelial rupture theory ……………………………………………… 23 1.5 Clinical management ………………………………………………………… 24 1.5.1 Symptoms ……………………………………………………………… 24 1.5.2 Diagnosis ………………………………………………………………… 24 1.5.3 Treatment ………………………………………………………………… 26 Chapter Objectives and Significance 2.1 Research questions ………………………………………………………… 31 2.2 Aims …………………………………………………………………………… 34 2.3 Significance …………………………………………………………………… 35 Chapter Materials and Methods 3.1 Study Subject (for superantigen and methylation studies) …………………… 36 3.2 Study Subject (for gene expression study) …………………………………… 37 3.3 Allergy test …………………………………………………………………… 38 3.4 DNA extraction ………………………………………………………………… 38 3.4.1 Extraction from solid tissues …………………………………………… 38 3.4.2 Extraction from peripheral blood mononuclear cell (PBMC) ………… 39 3.5 Experiments for superantigen study …………………………………………… 38 3.5.1 Standard polymerase chain reaction (PCR) ……………………………… 39 3.5.2 Direct sequencing ……………………………………………………… 40 vi 3.6 Experiments for methylation study …………………………………………… 41 3.6.1 Bisulfite modification of DNA …………………………………………… 41 3.6.2 Methylation-specific PCR (MSP) ……………………………………… 41 3.6.3 Bisulfite genomic sequencing (BGS) …………………………………… 44 3.7 Experiments for gene expression study ………………………………………… 44 3.7.1 RNA extraction from nasal tissues ……………………………………… 44 3.7.2 Quantification and gel electrophoresis of RNA ………………………… 45 3.7.3 Microarray experiment …………………………………………………… 45 3.7.4 Quality control (QC) assessment for microarray experiment and data … 47 3.7.5 Statistical analysis by Significant Analysis of Microrarray (SAM) ……… 51 3.7.6 Annotation analysis ……………………………………………………… 54 3.7.7 Class predictor analysis ………………………………………………… 54 3.7.8 Ingenuity Pathways Analysis (IPA) ……………………………………… 56 3.7.9 Real-time reverse transcription (RT) PCR ……………………………… 61 3.8 Histo-immunohistochemical examination …………………………………… 63 3.8.1 Staining procedures for frozen tissues …………………………………… 63 3.8.2 Staining procedures for paraffin embedded tissues ……………………… 64 3.8.3 Evaluation of histo-immunohistochemical patterns …………………… 65 3.9 Statistical analysis …………………………………………………………… 67 3.9.1 Statistics in methylation study ………………………………………… 67 3.9.2 Statistics in gene expression study ……………………………………… 67 Chapter Role of Staphylococcus Aureus and Superantigens in Nasal Polyposis Part I Results ……………………………………………………………………… 69 vii 4.1.1 Patient characteristics and histological evaluation ………………………… 69 4.1.2 Detection of S aureus and superantigens …………………………………… 69 Part II Discussion ………………………………………………………………… 72 Part III Conclusion ……………………………………………………………… 74 Chapter Methylation of tumor suppressor genes in Nasal Polyposis Part I Results ……………………………………………………………………… 75 5.1.1 Patient characteristics and histological evaluation ………………………… 75 5.1.2 Detection of methylation status by MSP …………………………………… 78 5.1.3 Confirmation of MSP results by BGS ……………………………………… 80 5.1.4 Correlation between methylation status and protein expression …………… 82 Part II Discussion ………………………………………………………………… 84 Part III Conclusion ……………………………………………………………… 88 Chapter Gene Expression Profiles in Nasal Polyposis and the Response of NP to Steroid Treatment Part I Results ……………………………………………………………………… 85 6.1.1 Patient characteristics and histological evaluation …………………………… 89 6.1.2 Strategy for identifying candidate genes by microarray analyses …………… 93 6.1.3 Quality of samples and array data …………………………………………… 96 6.1.4 Genome-wide transcriptional alterations …………………………………… 105 6.1.5 Classification of samples based on gene expression patterns …………… … 109 6.1.6 Functions of the significant genes ………………………………………… 114 6.1.7 Identification of GC-responsive genes by network analysis ………….…… 118 6.1.8 Identification of NP associated genes by Canonical Pathway analysis …… 126 viii 6.1.9 Identification of NP associated genes by histopathologic features ………… 141 6.1.10 Target genes validation by quantitative PCR ……………………………… 147 6.1.11 Protein expression evaluated by immunohistochemistry ………………… 150 Part II Discussion …………………………………………………………………153 6.2.1 Indication of microarray analysis …………………………………………… 154 6.2.2 Summary of the functional network pathways ……………………………… 158 6.2.3 Epithelial repair effect of GCs in NP …………………………………… 169 6.2.4 Anti-inflammatory effect of GCs in NP ….………….….……… … …… 175 6.2.5 Hypothesis of the GC beneficial effects on NP ….….….…….………….… 187 6.2.6 Combination of eosinophil- and neutrophil-infiltration in NP ……………… 189 6.2.7 Other gene families associated with pathogenesis of NP …………………… 198 Part III Conclusion ……………………………………………………………… 207 Chapter Conclusions and Suggested Future Studies 7.1 Summary of important findings ……………………………………………… 209 7.2 Limitation of the current study ……………………………………………… 210 7.3 Suggestions for future work ………………………………………………… 211 References ………………………………………………………… 215 Appendices Appendix I Significant functions of the datasets ………………………………… 265 Appendix II Fold change of interested genes in three datasets ………………… 268 Appendix III Relative expression level of selected genes by real-time RT PCR … 271 Curriculum Vitae ………………………………………………… 272 ix Summary Nasal polyposis (NP) is a chronic inflammatory airway disease, which represents severe infiltration of inflammatory cells (e.g eosinophils and neutrophils), epithelial damage, and stromal edema Although glucocorticosteroid (GC) treatment is effective in relieving NP inflammation, the high recurrence rate makes the etiology and pathogenesis of NP complicated The results from our research group reported profiles of cellular infiltration in Asian NP In this respect, this thesis focuses on the molecular mechanisms underlying the pathogenesis of Asian (especially Chinese) NP and its response to GC treatment At first, we started to test the hypothesis of Staphylococcus aureus (S aureus) and its superantigens in Asian NP A low incidence rate of S aureus was found in the studied NP and superantigens could not be found in all NP tissues, indicating no significant effects of S.aureus related superantigens in Asian NP Secondly, we tried to find if some cancer related mechanism (methylation) would be involved in NP pathogenesis This is based on the assumption that NP pathological features are somewhat similar to tumor growth, such as tissue hyperplasia and high recurrence rate Although methylation of common tumor suppressor genes (TSGs) (CDH1, TSLC1, DAPK1, and PTPN6) was detected in NP, the frequency of gene methylation did not differ between NP and nasal mucosal controls, indicating the role of methylation of these TSGs appears to be minimal in NP The first two studies came out with negative results which were not anticipated initially For this reason, a systemic microarray analysis was used to identify novel x of oral mucosal tissue injury and repair Wound Repair Regen 2005;13:19-26 Ward C, Chilvers ER, Lawson MF, Pryde JG, Fujihara S, Farrow SN, Haslett C, Rossi AG NF-kappaB activation is a critical regulator of human granulocyte apoptosis in vitro J Biol Chem 1999;274(7):4309-18 Watelet JB, Bachert C, Claeys C, Van Cauwenberge P.Matrix metalloproteinases MMP-7, MMP-9 and their tissue inhibitor TIMP-1: expression in chronic sinusitis vs nasal polyposis.Allergy 2004;59(1):54-60 Watelet JB, Claeys C, Perez-Novo C, Gevaert P, Van Cauwenberge P, Bachert C.Transforming growth factor beta1 in nasal remodeling: differences between chronic rhinosinusitis and nasal polyposis Am J Rhinol 2004;18(5):267-72 Watelet JB, Van Zele T, Gjomarkaj M, Canonica GW, Dahlen SE, Fokkens W, Lund VJ, Scadding GK, Mullol J, Papadopoulos N, Bonini S, Kowalski ML, Van Cauwenberge P, Bousquet J; GA(2)LEN Workpackage Members 2.7 Tissue remodelling in upper airways: where is the link with lower airway remodelling? Allergy 2006;61:1249-58 Weiler H, Lindner V, Kerlin B, Isermann BH, Hendrickson SB, Cooley BC, Meh DA, Mosesson MW, Shworak NW, Post MJ, Conway EM, Ulfman LH, von Andrian UH, Weitz JI.Characterization of a mouse model for thrombomodulin deficiency.Arterioscler Thromb Vasc Biol 2001;21(9):1531-7 Widney DP, Xia YR, Lusis AJ, Smith JB The murine chemokine CXCL11 (IFN-inducible T cell alpha chemoattractant) is an IFN-gamma- and lipopolysaccharide-inducible glucocorticoid-attenuated response gene expressed in lung and other tissues during endotoxemia J Immunol 2000;164(12):6322-31 Winoto A, Littman DR Nuclear hormone receptors in T lymphocytes Cell 2002;109 Suppl:S57-66 260 Wladislavosky-Waserman P, Kern EB, Holley KE, Eisenbrey AB, Gleich GJ Epithelial damage in nasal polyps Clin Allergy 1984;14:241-7 Woltmann G, McNulty CA, Dewson G, Symon FA, Wardlaw AJ Interleukin-13 induces PSGL-1/P-selectin-dependent adhesion of eosinophils, but not neutrophils, to human umbilical vein endothelial cells under flow.Blood 2000;95(10):3146-52 Wong CK, Zhang JP, Ip WK, Lam CW Activation of p38 mitogen-activated protein kinase and nuclear factor-kappaB in tumour necrosis factor-induced eotaxin release of human eosinophils Clin Exp Immunol 2002;128(3):483-9 Woodworth BA, Joseph K, Kaplan AP, Schlosser RJ.Alterations in eotaxin, monocyte chemoattractant protein-4, interleukin-5, and interleukin-13 after systemic steroid treatment for nasal polyps.Otolaryngol Head Neck Surg 2004;131(5):585-9 Wu CC, Croxtall JD, Perretti M, Bryant CE, Thiemermann C, Flower RJ, Vane JR.Lipocortin mediates the inhibition by dexamethasone of the induction by endotoxin of nitric oxide synthase in the rat.Proc Natl Acad Sci U S A 1995;92(8):3473-7 Xaubet A, Mullol J, Lopez E, Roca-Ferrer J, Rozman M, Carrion T, Fabra JM, Picado C.Comparison of the role of nasal polyp and normal nasal mucosal epithelial cells on in vitro eosinophil survival Mediation by GM-CSF and inhibition by dexamethasone.Clin Exp Allergy 1994;24(4):307-17 Yamada T, Fujieda S, Mori S, Yamamoto H, Saito H.Macrolide treatment decreased the size of nasal polyps and IL-8 levels in nasal lavage.Am J Rhinol 2000;14(3):143-8 Yamada T, Fujieda S, Yanagi S, Yamamura H, Inatome R, Sunaga H, Saito H.Protein-tyrosine kinase Syk expressed in human nasal fibroblasts and its effect on RANTES production.J Immunol 2001;166(1):538-43 261 Yamaguchi K, Lantowski A, Dannenberg AJ, Subbaramaiah K Histone deacetylase inhibitors suppress the induction of c-Jun and its target genes including COX-2 J Biol Chem 2005;280:32569-77 Yamashita T, Tsuji H, Maeda N, Tomoda K, Kumazawa T.Etiology of nasal polyps associated with aspirin-sensitive asthma.Rhinol Suppl 1989;8:15-24 Yamazaki S, Muta T, Takeshige K.A novel IkappaB protein, IkappaB-zeta, induced by proinflammatory stimuli, negatively regulates nuclear factor-kappaB in the nuclei.J Biol Chem 2001;276(29):27657-62 Yang-Yen HF, Chambard JC, Sun YL, Smeal T, Schmidt TJ, Drouin J, Karin M.Transcriptional interference between c-Jun and the glucocorticoid receptor: mutual inhibition of DNA binding due to direct protein-protein interaction Cell 1990; 62:1205-15 Yariktas M, Doner F, Sutcu R, Demirci M, Dogru H, Yasan H.The effect of topical corticosteroid on basic fibroblast growth factor in nasal polyp tissue.Am J Rhinol 2005;19(3):248-50 Yasuda M, Niisato N, Miyazaki H, Hama T, Dejima K, Hisa Y, Marunaka Y.Epithelial ion transport of human nasal polyp and paranasal sinus mucosa.Am J Respir Cell Mol Biol 2007b;36(4):466-72 Yasuda M, Niisato N, Miyazaki H, Iwasaki Y, Hama T, Dejima K, Hisa Y, Marunaka Y.Epithelial Na+ channel and ion transport in human nasal polyp and paranasal sinus mucosa.Biochem Biophys Res Commun 2007a;362(3):753-8 Yasunaga S, Yuyama N, Arima K, Tanaka H, Toda S, Maeda M, Matsui K, Goda C, Yang Q, Sugita Y, Nagai H, Izuhara K The negative-feedback regulation of the IL-13 signal by the IL-13 receptor alpha2 chain in bronchial epithelial cells Cytokine 2003;24(6):293-303 262 Yea SS, Yang YI, Park SK, Jang WH, Lee SS, Seog DH, Park YH, Chun JH Interleukin-4 C-590T polymorphism is associated with protection against nasal polyps in a Korean population Am J Rhinology 2006;20(5):550-3 Yin BW, Lloyd KO.Molecular cloning of the CA125 ovarian cancer antigen: identification as a new mucin, MUC16.J Biol Chem 2001;276(29):27371-5 Yoon HK, Cho HY, Kleeberger SR.Protective role of matrix metalloproteinase-9 in ozone-induced airway inflammation.Environ Health Perspect 2007;115(11):1557-63 Young Kim J, Kim CH, Kim KS, Choi YS, Lee JG, Yoon JH.Extracellular signal-regulated kinase is involved in tumor necrosis factor-alpha-induced MUC5AC gene expression in cultured human nasal polyp epithelial cells.Acta Otolaryngol 2004;124(8):953-7 Yusoff P, Lao DH, Ong SH, Wong ES, Lim J, Lo TL, Leong HF, Fong CW, Guy GR.Sprouty2 inhibits the Ras/MAP kinase pathway by inhibiting the activation of Raf.J Biol Chem 2002;277(5):3195-201 Zelko IN, Mariani TJ, Folz RJ.Superoxide dismutase multigene family: a comparison of the CuZn-SOD (SOD1), Mn-SOD (SOD2), and EC-SOD (SOD3) gene structures, evolution, and expression.Free Radic Biol Med 2002;33(3):337-49 Zhang N, Holtappels G, Claeys C, Huang G, van Cauwenberge P, Bachert C.Pattern of inflammation and impact of Staphylococcus aureus enterotoxins in nasal polyps from southern China.Am J Rhinol 2006 ;20(4):445-50 Zhang S, Smartt H, Holgate ST, Roche WR Growth factors secreted by bronchial epithelial cells control myofibroblast proliferation: an in vitro co-culture model of airway remodeling in asthma Lab Invest 1999;79(4):395-405 263 Zheng CF, Guan KL.Dephosphorylation and inactivation of the mitogen-activated protein kinase by a mitogen-induced Thr/Tyr protein phosphatase.J Biol Chem 1993;268(22):16116-9 Zheng Z, Pan J, Chu B, Wong YC, Cheung AL, Tsao SW Downregulation and abnormal expression of E-cadherin and beta-catenin in nasopharyngeal carcinoma: close association with advanced disease stage and lymph node metastasis Hum Pathol 1999;30:458-466 Zídková J, Stĕdrý V, Zídek V, Volfová I, Síbl O.Participation of complement in nasal polyposis.Folia Microbiol (Praha) 1993;38(2):150-2 264 Appendices Appendix I Significant functions of the datasets GC-naïve NP versus control Significance p-value* Genes [n] Cancer 2.82E-25 to 1.26E-03 554 Developmental Disorder 2.08E-11 to 3.46E-04 135 Inflammatory Disease 1.30E-09 to 1.26E-03 188 Connective Tissue Disorders 3.59E-09 to 8.06E-04 168 Cardiovascular Disease 1.51E-08 to 1.26E-03 130 Immunological Disease 6.29E-06 to 1.21E-03 152 Respiratory Disease 1.47E-05 to 1.26E-03 112 Organismal Injury and Abnormalities 4.73E-05 to 8.01E-04 61 Genetic Disorder 4.86E-05 to 1.26E-03 160 Tissue Development 2.59E-15 to 1.26E-03 270 Organismal Development 3.70E-12 to 5.66E-04 210 Cardiovascular System Development and Function 6.97E-12 to 1.26E-03 154 Tissue Morphology 4.39E-08 to 1.26E-03 227 Organismal Survival 1.51E-06 to 5.82E-06 140 Organ Development 8.95E-06 to 7.63E-04 145 Tumor Morphology 1.47E-05 to 1.26E-03 66 Immune Response 2.20E-05 to 9.57E-04 106 Connective Tissue Development and Function 2.96E-05 to 1.26E-03 142 Organismal Functions 2.45E-04 to 2.45E-04 19 Cellular Movement 6.51E-15 to 8.89E-04 282 Cellular Growth & Proliferation 9.88E-13 to 1.24E-03 456 Cell Death 6.74E-12 to 1.19E-03 411 Cellular Development 1.41E-10 to 1.26E-03 336 Cell morphology 1.56E-08 to 1.26E-03 230 Cell Cycle 4.14E-09 to 1.26E-03 197 Cellular Assembly and Organization 9.50E-08 to 1.26E-03 155 Gene Expression 1.80E-06 to 1.26E-03 268 Cell-to-Cell Signaling and Interaction 1.94E-06 to 6.87E-04 122 Cellular Function and Maintenance 2.26E-04 to 1.21E-03 45 Category Diseases & Disorders Physiological System Development & Function Molecular & Cellular Functions 265 GC-treated NP versus control Significance p-value* Genes [n] Cancer 2.14E-12 to 1.38E-02 317 Developmental Disorder 2.83E-11 to 9.51E-03 98 Cardiovascular Disease 8.28E-06 to 1.20E-02 72 Category Diseases & Disorders Genetic Disorder 4.57E-05 to 1.38E-02 120 Respiratory Disease 3.36E-04 to 1.38E-02 60 Organismal Injury and Abnormalities 1.27E-03 to 1.38E-02 30 Inflammatory Disease 4.73E-03 to 1.00E-02 101 Immunological Disease 5.18E-03 to 5.18E-03 Tissue Morphology 1.03E-08 to 1.18E-02 75 Tissue Development 1.07E-06 to 1.38E-02 165 Cardiovascular System Development and Function 1.03E-04 to 1.38E-02 91 Organismal Development 1.23E-04 to 1.29E-02 122 Organ Development 2.22E-04 to 1.38E-02 132 Immune Response 3.03E-04 to 1.38E-02 65 Immune and Lymphatic System Development 3.03E-04 to 1.38E-02 75 Organ Morphology 7.93E-04 to 1.38E-02 48 Connective Tissue Development and Function 1.01E-03 to 1.18E-02 64 Tumor Morphology 1.55E-03 to 1.33E-02 22 Cellular Movement 6.23E-08 to 1.38E-02 176 Cell Morphology 8.00E-06 to 1.38E-02 151 Cellular Assembly and Organization 2.92E-05 to 1.38E-02 112 Cell Signaling 3.38E-05 to 1.38E-02 73 Cellular Growth & Proliferation 5.00E-05 to 1.38E-02 266 Cellular Development 6.93E-05 to 1.38E-02 190 Cell-to-Cell Signaling and Interaction 7.56E-05 to 1.38E-02 101 Lipid Metabolism 1.1E-04 to 9.51E-03 15 Small Molecule Biochemistry 1.1E-04 to 1.38E-02 64 Cell Death 1.30E-04 to 1.33E-02 241 Physiological System Development & Function Molecular & Cellular Functions 266 GC-treated versus steroid naïve NP Significance p-value* Genes [n] Inflammatory Disease 5.43E-15 to 1.86E-04 33 Cancer 1.24E-12 to 2.58E-04 51 Cardiovascular Disease 2.37E-12 to 1.73E-05 23 Connective Tissue Disorders 8.03E-12 to 1.86E-04 33 Immunological Disease 1.78E-10 to 1.23E-04 29 Organismal Injury and Abnormalities 1.47E-08 to 2.39E-04 16 Category Diseases & Disorders Genetic Disorder 9.19E-07 to 9.19E-07 Respiratory Disease 3.44E-05 to 1.15E-04 Developmental Disorder 1.10E-04 to 1.10E-04 14 Organismal Functions 6.90E-10 to 1.27E-05 Tissue Morphology 9.30E-10 to 2.28E-04 26 Tissue Development 2.24E-08 to 2.32E-04 34 Immune Response 3.54E-08 to 1.55E-04 29 Organismal Development 6.69E-08 to 2.77E-05 25 Tumor Morphology 2.67E-07 to 2.28E-04 11 Organ Morphology 9.54E-07 to 8.06E-05 Connective Tissue Development and Function 1.09E-06 to 2.46E-04 23 Organ Development 1.13E-06 to 1.13E-06 18 Cardiovascular System Development and Function 1.20E-06 to 2.52E-04 19 Cell Death 1.83E-14 to2.15E-04 47 Cellular Growth & Proliferation 1.08E-13 to 2.41E-04 53 Cellular Development 9.50E-13 to 2.46E-04 41 Cellular Movement 1.59E-12 to 2.52E-04 37 Cell Cycle 3.63E-09 to 2.58E-04 25 Gene Expression 3.94E-09 to 2.58E-04 31 Cell-to-Cell Signaling and Interaction 2.15E-08 to 2.28E-04 27 DNA Replication, Recombination, and Repair 1.38E-06 to 1.38E-06 13 Cell Morphology 1.68E-06 to 1.82E-04 22 Cellular Function and Maintenance 3.84E-05 to 2.28E-04 Physiological System Development & Function Molecular & Cellular Functions * p-value is determined by right-tailed Fischer’s exact test 267 Appendix II Fold change of interested genes in three datasets (measured by microarray and quantitative PCR) Fold difference assessed by means of microarray GC-naïve GC-treated NP vs NP vs control control ADAM8 1.61 NS ALOX5AP 2.61 NS ANGPT1 0.27 0.29 ANGPT2 0.34 0.36 ANXA1 NS NS AREG 0.22 NS ATP1A2 0.34 0.35 Bid 1.65 NS C1QB 4.73 2.67 C3 2.64 2.21 C4A 5.54 3.97 CASP3 1.73 1.60 CASP7 1.85 1.70 CCL11 3.87 NS CCL15 4.68 3.76 CCL28 0.18 0.22 CD40 0.51 NS CD69 0.41 NS CD86 3.39 2.38 CD9 0.51 NS CEACAM1 0.57 NS CEACAM6 6.06 4.68 CFH 0.49 NS c-Fos 0.08 NS c-Jun 0.29 NS CLIC3 2.03 NS CLIC5 1.85 NS CLIC6 3.57 4.41 COX-2 0.38 NS CRISP3 0.01 0.14 CXCL11 NS NS CXCL12 0.34 0.32 CXCL2 0.61 NS CXCL6 7.65 6.09 CXCL9 NS NS Gene symbol GC-treated vs GC -naïve NP NS NS NS NS 5.91 6.47 NS NS NS NS NS NS NS NS NS NS NS 2.99 NS NS NS NS NS 14.85 3.39 NS NS NS 3.47 NS 0.4 NS 5.42 NS 0.4 Fold difference assessed by means of quantitative PCR GC-naïve GC-treated GC-treated NP vs NP vs vs GC control control -naïve NP N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A 4.55 0.01 NS 6.7 N.A N.A N.A N.A N.A N.A N.A N.A N.A 5.75 N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A 14.92 NS N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A 0.05 NS N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A 0.02 NS 14.2 0.06 NS 4.9 N.A N.A N.A N.A N.A N.A N.A N.A N.A 0.17 NS 5.51 N.A N.A N.A N.A N.A 0.35 N.A N.A N.A 0.14 NS 7.45 N.A N.A N.A N.A N.A 0.4 268 Fold difference assessed by means of microarray Gene symbol CYSLTR1 DEFB1 DUOX1 DUSP1 DUSP2 DUSP4 DUSP5 DUSP6 EGF EGR1 ERBB4 FosB GCLM GPX3 GRα GRβ HBEGF IFNAR1 IL13RA2 IL18 IL5Ra IL-6 IL6ST ITGB2 JunB LGALS8 LGALS9 LPO LTA4H LTB4R LYN MIF MMP7 MMP9 MUC16 MUC20 MUC4 MUC7 Fold difference assessed by means of quantitative PCR GC-naïve GC-treated NP vs NP vs control control 2.92 3.05 3.93 2.53 6.67 2.56 0.1 NS 0.18 NS 0.21 0.32 0.22 NS 0.34 NS 0.25 NS 0.08 0.36 0.47 NS 0.05 NS 3.65 3.3 0.38 NS N.A N.A N.A N.A 0.21 NS 1.56 NS 0.49 NS 3.03 2.9 1.58 NS 0.16 0.42 0.59 NS 2.62 NS 0.24 0.37 1.64 NS 1.69 NS 0.03 0.23 1.53 NS 1.66 2.35 1.97 NS 1.75 NS 6.43 2.48 3.48 NS 11.12 11.21 4.51 3.58 5.47 5.48 0.02 0.06 GC-naïve GC-treated GC-treated NP vs NP vs vs GC control control -naïve NP N.A N.A N.A N.A N.A N.A N.A N.A N.A 0.12 NS 8.35 0.06 NS 2.8 N.A N.A N.A N.A N.A N.A 0.16 NS 2.5 N.A N.A N.A 0.01 0.12 12.3 N.A N.A N.A 0.01 NS 15.3 N.A N.A N.A N.A N.A N.A 0.31 0.34 1.2 UDab UDab UDab 0.06 NS 5.05 N.A N.A N.A UDa N.A N.A 4.37 N.A N.A 9.85 NS N.A 0.01 0.2 9.85 N.A N.A N.A N.A N.A N.A 0.06 0.15 3.15 N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A 8.62 4.05 0.5 UDb UDb 0.45 N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A GC-treated vs GC -naïve NP NS NS NS 4.98 3.19 NS NS 2.08 NS 7.81 NS 16.67 NS 1.59 N.A N.A 5.45 NS NS NS NS 7.87 NS NS 3.37 NS NS NS NS NS NS NS 0.47 0.44 NS NS NS NS 269 Fold difference assessed by means of microarray Gene symbol NFKBIA NFKBIZ NOS2A NOX4 NR4A1 NR4A2 NR4A3 NRG3 OXR1 PLA2G10 PLA2G4A PRDX1 PRDX5 PTGER2 PTGER3 PTGIS PTX3 SCGB1A1 SCNN1A SCNN1B SCNN1G SELPLG SERPINA1 SOCS3 SOD3 SPRY1 SPRY2 SPRY4 STAT3 TEK THBD TXN ZFP36 Fold difference assessed by means of quantitative PCR GC-naïve GC-treated NP vs NP vs control control 0.65 NS 0.46 NS 3.96 6.76 3.03 NS 0.14 NS 0.11 NS 0.11 0.28 0.23 0.29 0.42 0.39 2.51 2.53 2.61 3.28 1.62 NS 1.79 NS 1.99 NS 0.15 0.29 0.26 0.25 0.12 NS NS NS 1.96 2.11 4.93 6.04 5.65 7.34 2.04 NS 3.18 3.48 0.31 NS 0.39 0.47 0.33 NS 0.62 NS 0.62 NS 0.58 NS 0.31 0.41 0.22 NS 1.64 NS 0.17 NS GC-naïve GC-treated GC-treated NP vs NP vs vs GC control control -naïve NP 0.45 N.A N.A 0.23 NS 2.2 N.A N.A N.A N.A N.A N.A 0.03 NS 9.15 0.08 NS 6.25 N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A 4.9 N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A 0.21 NS 3.8 N.A N.A N.A 0.19 NS 2.1 0.51 NS 2.21 0.42 NS 2.03 N.A N.A N.A N.A N.A N.A 0.09 NS 2.7 N.A N.A N.A 0.11 NS 5.35 GC-treated vs GC -naïve NP NS 2.22 NS NS 4.36 7.02 NS NS NS NS NS NS NS NS NS NS NS 5.51 NS NS NS NS NS 4.2 NS 2.49 2.11 2.13 NS NS 2.63 NS 3.77 NS, No statistical difference; UDa, Undetectable in NP (either GC-naïve or GC-treated); Udb, Undectable in control; Udab, Undectable in both NP and control; N.A., Not applicable 270 Appendix III Relative expression level of selected genes by real-time RT PCR Median of ∆ Ct (Ct-target – Ct-GAPDH) Gene symbol ANXA1 AREG C3 CCL11 CD69 c-Fos c-Jun COX-2 CXCL11 CXCL2 CXCL9 DUSP1 DUSP2 DUSP6 EGR1 FosB GRα GRβ HBEGF IFNAR1 IL13RA2 IL18 IL5Ra IL-6 JunB MMP7 MMP9 NFKBIA NFKBIZ NR4A1 NR4A2 SCGB1A1 SOCS3 SPRY1 THBD ZFP36 GC-naïve NP (n=10) 5.45 12.85 2.15 5.56 11.94 5.74 7.67 9.26 8.53 8.75 5.45 4.52 11.98 5.75 5.31 10.59 8.10 UD 8.45 N.A UD 10.37 9.67 13.24 5.19 6.41 5.89 3.40 7.85 7.43 7.82 11.72 5.68 9.79 8.45 5.09 GC-treated NP (n=10) 3.50 8.98 N.A N.A 9.46 0.97 5.26 6.8 9.8 6.35 6.32 1.00 9.92 4.57 1.86 5.31 7.97 UD 5.66 N.A UD N.A N.A 9.12 3.98 7.51 7.33 N.A 6.23 3.26 5.19 9.29 4.05 9.15 7.09 3.21 Control (n=6) N.A 6.59 4.68 9.52 7.59 0.13 3.6 6.72 N.A 5.67 N.A 1.47 7.97 3.06 -0.56 4.52 6.41 UD 4.61 N.A UD 12.36 12.22 6.82 1.01 9.18 UD 2.18 5.67 2.19 4.09 11.99 3.40 7.47 5.05 1.88 UD, Undetectable N.A., Not applicable 271 Curriculum Vitae Name: Li Chunwei Gender: Male Birthday: March 12th 1979 Nationality: Chinese Email: lichunwei@nus.edu.sg Mobile phone: 65-91162358 Address: Department of Otolaryngology, National University of Singapore, Lower Kent Ridge Road Singapore, 119074 Education 01/2003-Present PhD candidature in Department of Otolaryngology, National University of Singapore, Singapore 09/1997-07/2002 Bachelor of Medicine in Sun Yat-sen University, P.R China Working Experience 01/2007-Present Research assistant in Department of Otolaryngology, National University of Singapore, Singapore 02/2002-07/2002 Internship in the Center of Disease Control in Guangzhou, P.R China 09/2001-02/2002 Internship in the Faculty of public health of Sun Yat-sen University, P.R China 11/2000-08/2001 Internship in The First affiliated hospital of Sun Yet-sen University, P.R China 272 Scholarship and Award 01/2003-01/2007 NUS Research Scholarship, National University of Singapore, Singapore 06/2005 Travel grant of 2005 Wordl Allergy Congress, Munich, Germany 06/2008 Travel grant of XXVII Congress of the European Academy of Allergology and Clinical Immunology, EAACI 2008, Barcelona, Spain Membership 01/2005-Present Junior member of European Academy of Allergology and Clinical Immunology Skills profile Be familiar with molecular and cell biology techniques, such as DNA/RNA purification, microarray, Real-time PCR, immunohistochemistry and cell culture work Be familiar with biostatistics work and related software 273 Publications Li CW, Cheung W, Lin ZB, Li TY, Lim JT, Wang DY Oral steroids enhance epithelial repair in nasal polyposis via up-regulation of AP-1 gene network Thorax 2009 Jan 21 [Epub ahead of print] (Impaction Factor: 7.06 Liang XH, Cheung W, Heng CK, Liu JJ, Li CW, Lim B, Wang DY CD14 promoter polymorphisms have no functional significance and are not associated with atopic phenotypes Pharmacogenet Genomics 2006 Apr;16(4):229-36 (Impaction Factor: 4.14) Wang DY, Li CW Control of nasal obstruction in patients with persistent allergic rhinitis Zhonghua Er Bi Yan Hou Tou Jing Wai Ke Za Zhi 2006 Sep;41(9):716-20 Li CW, Cheung W, Li TY, Lin ZB, Lim JT, Wang DY Expression profile of eosinophil- and neutrophil-associated genes in patients with nasal polyposis Article submitted Li CW, Cheung W, Pang YT, Wang DY Low level methylation of some tumor suppressor genes in nasal polyps and normal nasal mucosa Article in preparation Presentations at conferences Li CW, Pang YT, Tao Q, Wang DY Promoter methylation status of multiple genes in nasal polyps Poster presentation in 2005 World Allergy Congress, Munich, Germany Poster No 751 Li CW, Cheung W, Lin ZB, Li TY, Lim JT, Wang DY Glucocorticoids promote epithelial repair in nasal polyps via upregulating Activator protein-1 Oral presentation in XXVII Congress of the European Academy of Allergology and Clinical Immunology, EAACI 2008, Barcelona, Spain Abstract No 119 (Awarded with the best oral presentation in the session of “Inflammatory Mechanisms in Rhinosinusal Disease”.) 274 ... in the session of “Inflammatory Mechanisms in Rhinosinusal Disease”.) iv Molecular mechanisms underlying the pathogenesis of nasal polyposis and its response to steroid treatment Content of the. .. through the epithelial defect and the mucosa tends to cover it by migration of the epithelium from the edges 23 of the defect; (iii) the prolapse of the lamina propria undergoes epithelialization, and. .. demonstrate the molecular profiles underlying the beneficial effects of GCs on NP and the histopathological patterns of NP Identification of these genes and gene networks ultimately contributes to the knowledge