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published_or_final_version / abstract / Surgery / Doctoral / Doctor of Philosophy

published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

鼻咽癌(nasopharyngeal carcinoma, NPC)與艾巴氏病毒(Epstein-Barr virus, EBV)和遺傳及表現遺傳變異有連串關係。儘管鼻咽癌腫瘤的發生機制仍然未知,最近研究顯示微核糖核酸(microRNA, miRNA)通過調節細胞增殖凋亡遷移和侵襲等方式對鼻咽癌的生成起著至關重要的作用。為了確定微核糖核酸與鼻咽癌發生的相關機制及其扮演的角色，我們集中研究微核糖核酸在鼻咽癌腫瘤中所發生的變異，探討這些異常表達的微核糖核酸的功能，並揭開與幹細胞相關的微核糖核酸在鼻咽癌幹細胞樣細胞(cancer stem-like cell, CSC)裏所扮演的角色。 / 通過使用微陣列技術(Agilent Microarray)， 我們運用了 866個人類與 89個病毒微核糖核酸探針,以識別出多個帶有艾巴氏病毒的鼻咽癌腫瘤細胞系裏的微核糖核酸表達圖譜。相比正常的鼻咽上皮細胞系NP69，113個微核糖核酸在鼻咽癌中的差異表達已被鑒定出來。其中58個在鼻咽癌裏下調的微核糖核酸表達，miR-31的轉錄下調現象在鼻咽癌腫瘤細胞系和原發腫瘤中被不斷地發現。在7個帶有艾巴氏病毒的腫瘤細胞系樣本裏, 其中6個(86%)樣本呈miR-31下調跡象。與此同時，以顯微切割技術所得的38個原發腫瘤樣本中全部(100%)都顯示有miR-31下調的跡象。相比之下，所有正常的鼻咽上皮細胞都顯出高表達的miR-31。 / miR-31位於染色體9p21.3上，距離CDKN2A (p16) 0.5Mb處。這是在鼻咽癌細胞裏通常缺失的位置。在X1915和X99186腫瘤細胞系中，已證實在miR-31和CDKN2A位點上都出現了純合性缺失。在四株不具備miR-31缺失的腫瘤細胞系裏，甲基化特異性聚合酶連鎖反應 (methylation-specific PCR, MSP) 和亞硫酸氫鈉測序法(bisulfite sequencing)發現了高甲基化的CpG島。使用5-aza-2’-deoxycytidine (5-Aza-dC) 治療後，鼻咽癌細胞株C666-1被證實恢復了miR-31轉錄。這些結果表明，純合性缺失和啟動子高甲基化是造成miR-31在鼻咽癌裏轉錄失效的主要發生機制。 / 微陣列技術和生物信息學分析找出了一些可能受miR-31影響的基因。其中FIH1和MCM2被確定為在鼻咽癌細胞裏受miR-31影響的基因。我們證實miR-31與FIH1和MCM2 信使核糖核酸的3’UTR處結合會抑制螢光素酶的活性。在鼻咽癌細胞裏miR-31的異位表達也會壓抑FIH1和MCM2蛋白的表達。更重要的是，恢復正常的miR-31表達或敲除FIH1表達能顯著地抑制C666-1細胞的增殖和移動能力。C666-1細胞的克隆形成能力和錨定依賴性生長都顯著地被miR-31的表達所抑制。穩定的miR-31表達亦能抑制鼻咽癌腫瘤在裸鼠體內的生長。此外，FIH1的敲除加強了p21和磷酸化p53 (Ser15) 的表達。這些結果暗示了miR-31是一個與鼻咽癌至關重要的微核糖核酸。 它通過了對FIH1的壓制，負面地調節細胞的增殖和移動。 / 使用微核糖核酸微陣列分析後，我們在鼻咽癌細胞中培養的懸浮細胞球裏篩選出差異表達的微核糖核酸。同樣地，實時螢光定量逆轉錄聚合酶鏈反應(qRT-PCR) 亦證實了miR-96和miR-183在C666-1懸浮細胞球裏是被抑制的。此外，miR-96和miR-183的異位表達顯著地降低了C666-1懸浮細胞球形成和克隆形成的能力。這項研究結果暗示, miR-96和miR-183的抑制對鼻咽癌幹細胞樣細胞的形成非常重要。 / 總的來說，某些微核糖核酸已被確定為潛在的鼻咽癌腫瘤抑制基因。 在帶艾巴氏病毒的鼻咽癌裏，miR-31的表達被證實是因純合性缺失和啟動子高甲基化而被下調的。miR-31抑制鼻咽癌細胞的增殖錨定依賴性生長細胞遷移和體內腫瘤的生長。同時，miR-96和miR-183也被發現對維持鼻咽癌的幹細胞樣特性起著一定作用。這些結果表明微核糖核酸對鼻咽癌腫瘤的生成扮演著抑制的角色。對微核糖核酸的機制作進一步全面了解將改進鼻咽癌的治療策略。 / Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) has been reported to be related to a number of genetic and epigenetic changes, however, the molecular mechanism leading to NPC tumorigenesis still remains unclear. Recently, microRNAs (miRNAs) have been demonstrated to play vital roles in NPC development via regulating cell proliferation, apoptosis, and cell migration and invasion. In this study, we aim to elucidate the role of miRNAs in NPC tumorigenesis in this study by identifying the miRNA aberration, investigating the possible functions of these aberrantly expressed miRNAs, and unraveling the role of stemness-related miRNAs in NPC cancer stem-like cells (CSCs). / By using Agilent Microarray with 866 human and 89 viral miRNA probes, miRNA expression profiles of multiple EBV-associated NPC tumor lines were generated. Compared to NP69, a nonmalignant nasopharyngeal epithelial cell line, 113 differentially expressed miRNAs were identified. Among the 58 down-regulated miRNAs in NPC, transcriptional silencing of miR-31 was consistently found in both NPC tumor lines and primary tumors. Down-regulation of miR-31 was detected in 6 of 7 (86%) EBV-positive tumor lines and 38 of 38 (100%) microdissected primary tumors, while all normal nasopharyngeal epithelia showed high expression of miR-31. / miR-31 is located at 0.5 Mb telomeric to CDKN2A (p16) on chromosome 9p21.3, which is commonly deleted in NPC. Homozygous deletion of both miR-31 and CDKN2A loci was confirmed in tumor lines X1915 and X99186. In the four tumor lines with intact miR-31, hypermethylation of 5’ CpG islands was detected by methylation-specific PCR (MSP) and bisulfite sequencing analysis. Restoration of miR-31 transcription was demonstrated in the EBV-positive NPC cell line C666-1 treated with 5-aza-2’-deoxycytidine. These findings suggested that homozygous deletion and promoter hypermethylation are the major mechanisms for transcriptional silencing of miR-31 in NPC. / By microarray and bioinformatic analysis, a number of putative targets of miR-31 were identified. Among these candidates, FIH1 and MCM2 were found to be the targets of miR-31 in NPC. We have shown that binding of miR-31 on FIH1 and MCM2 mRNA 3’UTR suppressed their luciferase activity. Ectopic expression of miR-31 in NPC cells resulted in repression of FIH1 and MCM2 protein expression. Importantly, the restoration of miR-31 or knockdown of FIH1 expression significantly suppressed proliferation as well as migration of C666-1 cells. Clone-forming ability and anchorage-independent growth of C666-1 were significantly inhibited by miR-31 expression. Stably expressed miR-31 was also demonstrated to inhibit NPC tumor growth in nude mice. Furthermore, expression of p21 and phospho-p53 (Ser15) was found to be increased by FIH1 knockdown. These results implied that miR-31 is a critical NPC-associated miRNA which negatively regulates cell proliferation and migration via FIH1 repression. / By miRNA microarray analysis, we have screened for differentially expressed miRNAs in sphere-forming cells of EBV-associated NPC. In concordance with microarray findings, suppression of miR-96 and miR-183 in C666-1 spheroids was confirmed by qRT-PCR. Ectopic expression of miR-96 and miR-183 significantly reduced the sphere-forming and clone-forming ability of C666-1 cells. The findings implied that miR-96 and miR-183 repression is important in the formation of NPC CSCs. / In summary, several miRNAs were identified as potential tumor suppressor genes in NPC. miR-31 was found down-regulated by homozygous deletion or promoter hypermethylation in EBV-associated NPC. It plays roles in NPC pathogenesis by suppressing NPC cell proliferation, clone-forming ability, cell anchorage-independent growth, migration and in vivo tumor growth. Moreover, miR-96 and miR-183 were found to have a role in the maintenance of NPC stem-like properties. These findings suggested important tumor suppressive roles of miRNAs in regulating NPC tumorigenesis, and a better understanding on the miRNA mechanisms may potentiate better therapeutic strategies for NPC. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Cheung, Ching Mei. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 177-209). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese. / Abstract --- p.i / 摘要 --- p.iv / Thesis / Assessment Committee --- p.vii / Acknowledgements --- p.viii / Table of contents --- p.ix / List of figures --- p.xv / List of tables --- p.xviii / List of publications --- p.xix / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Nasopharyngeal carcinoma (NPC) --- p.1 / Chapter 1.1.1 --- Histopathology and epidemiology --- p.1 / Chapter 1.1.2 --- Etiology --- p.2 / Chapter 1.1.2.1 --- Environmental factors --- p.2 / Chapter 1.1.2.2 --- Genetic factors --- p.3 / Chapter 1.1.2.3 --- Epstein-Barr virus (EBV) infection --- p.3 / Chapter 1.2 --- Molecular pathogenesis of NPC --- p.5 / Chapter 1.2.1 --- Cytogenetic changes --- p.5 / Chapter 1.2.2 --- NPC-associated tumor suppressor genes (TSGs) --- p.6 / Chapter 1.2.3 --- NPC-associated oncogenes --- p.8 / Chapter 1.3 --- MicroRNAs --- p.10 / Chapter 1.3.1 --- Biogenesis of microRNAs --- p.10 / Chapter 1.3.2 --- MicroRNAs and cancers --- p.15 / Chapter 1.3.2.1 --- MicroRNAs - tumor suppressors --- p.15 / Chapter 1.3.2.2 --- MicroRNAs - oncogenes --- p.16 / Chapter 1.4 --- MicroRNAs in nasopharyngeal carcinoma --- p.18 / Chapter 1.4.1 --- MicroRNA profiling in NPC --- p.18 / Chapter 1.4.2 --- OncomiRs in NPC --- p.20 / Chapter 1.4.3 --- Tumor suppressor miRNAs in NPC --- p.22 / Chapter 1.4.4 --- miRNAs and cancer stem-like cells (CSCs) --- p.27 / Chapter 1.4.5 --- Clinical implication of miRNAs in NPC --- p.29 / Chapter 1.5 --- Aims of study --- p.32 / Chapter Chapter 2 --- Materials and methods --- p.34 / Chapter 2.1 --- Patient biopsies --- p.34 / Chapter 2.2 --- NPC cell lines and xenografts --- p.34 / Chapter 2.2.1 --- Cell lines --- p.34 / Chapter 2.2.2 --- Xenografts --- p.36 / Chapter 2.3 --- Total RNA Isolation --- p.39 / Chapter 2.4 --- DNA extraction --- p.39 / Chapter 2.5 --- Protein Extraction --- p.40 / Chapter 2.6 --- Western Blotting --- p.40 / Chapter 2.7 --- Microarray analysis --- p.43 / Chapter 2.7.1 --- MicroRNA microarray --- p.43 / Chapter 2.7.2 --- Gene expression microarray --- p.44 / Chapter 2.8 --- Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) --- p.45 / Chapter 2.8.1 --- Conventional qRT-PCR --- p.45 / Chapter 2.8.2 --- Stem-looped qRT-PCR --- p.46 / Chapter 2.9 --- Preparation of stable clone of miR-31 --- p.51 / Chapter 2.9.1 --- Cloning and plasmid DNA preparation --- p.51 / Chapter 2.9.1.1 --- Bacterial Transformation --- p.51 / Chapter 2.9.1.2 --- Plasmid DNA Extraction --- p.51 / Chapter 2.9.2 --- DNA Sequencing --- p.52 / Chapter 2.9.3 --- Stable transfection --- p.52 / Chapter 2.9.4 --- Clone selection --- p.53 / Chapter 2.10 --- Transient transfection --- p.55 / Chapter 2.11 --- Flow cytometry --- p.55 / Chapter 2.11.1 --- Apoptosis analysis by Annexin V --- p.55 / Chapter 2.11.2 --- Cell cycle analysis by propidium iodide (PI) --- p.56 / Chapter 2.11.3 --- Detection of stem-like cell markers --- p.56 / Chapter 2.12 --- Cell proliferation analysis --- p.56 / Chapter 2.12.1 --- WST-1 assay --- p.56 / Chapter 2.12.2 --- BrdU assay --- p.57 / Chapter 2.13 --- Anchorage-independent growth assay --- p.58 / Chapter 2.14 --- Clone formation assay --- p.58 / Chapter 2.15 --- Cell migration assay --- p.54 / Chapter 2.16 --- In vivo tumorigenicity --- p.59 / Chapter 2.17 --- Dual luciferase reporter assay --- p.60 / Chapter 2.17.1 --- Luciferase reporter vectors --- p.60 / Chapter 2.17.2 --- Luciferase reporter assay --- p.60 / Chapter 2.18 --- Mapping homozygous deletion and genes in chromosome 9p21.3 --- p.64 / Chapter 2.19 --- 5-aza-2’-deoxycytidine (5-Aza-dC) and Trichostatin A (TSA) treatments --- p.64 / Chapter 2.20 --- Methylation specific-PCR (MSP) and bisulfite sequencing analysis --- p.68 / Chapter 2.20.1 --- Bisulfite modification --- p.68 / Chapter 2.20.2 --- Methylation specific-PCR (MSP) --- p.69 / Chapter 2.20.3 --- Bisulfite sequencing analysis --- p.69 / Chapter 2.21 --- Statistical analysis --- p.70 / Chapter 2.22 --- In situ hybridization (ISH) analysis --- p.73 / Chapter Chapter 3 --- Identification of novel deregulated microRNAs in nasopharyngeal carcinoma --- p.74 / Chapter 3.1 --- Introduction --- p.74 / Chapter 3.2 --- Results --- p.80 / Chapter 3.2.1 --- Aberrant expression of microRNAs in NPC --- p.80 / Chapter 3.2.2 --- Homozygous deletion of miR-31 in NPC --- p.90 / Chapter 3.2.3 --- Hypermethylation of 5’ CpG islands of miR-31 in NPC --- p.92 / Chapter 3.2.4 --- Detection of miR-31 expression in normal epithelia and NPC by in situ hybridization --- p.99 / Chapter 3.3 --- Discussion --- p.101 / Chapter Chapter 4 --- Characteristics of miR-31 and its targets in NPC --- p.105 / Chapter 4.1 --- Introduction --- p.105 / Chapter 4.2 --- Results --- p.107 / Chapter 4.2.1 --- Effects of exogenous miR-31 on NPC cells --- p.107 / Chapter 4.2.1.1 --- miR-31 effect on C666-1 cell proliferation and cell cycle progression --- p.107 / Chapter 4.2.1.2 --- Clone-forming ability and anchorage-independent growth of C666-1 --- p.113 / Chapter 4.2.1.3 --- Migration ability of C666-1 --- p.113 / Chapter 4.2.2 --- Effects of stably expressed miR-31 on NPC cells --- p.117 / Chapter 4.2.2.1 --- Stable clones selection by restoring precursor of miR-31 into C666-1 --- p.117 / Chapter 4.2.2.2 --- Cell proliferation and cell cycle progression in stable clones of miR-31 --- p.117 / Chapter 4.2.2.3 --- Anchorage-independent growth of C666-1 stable clones --- p.117 / Chapter 4.2.2.4 --- Tumorigenicity of C666-1 stable clones expressing miR-31 in vivo --- p.118 / Chapter 4.2.3 --- Identification of miR-31 targets in NPC cells --- p.125 / Chapter 4.2.3.1 --- miR-31 targets FIH1 and MCM2 --- p.125 / Chapter 4.2.3.2 --- Other reported targets of miR-31 in NPC --- p.131 / Chapter 4.2.4 --- Functional analysis of FIH1 in NPC cells --- p.133 / Chapter 4.2.4.1 --- Repression of FIH1 by siRNAs --- p.133 / Chapter 4.2.4.2 --- Proliferation of C666-1 with FIH1 knockdown --- p.133 / Chapter 4.2.4.3 --- Clone-forming and migration ability of C666-1 transfected with siFIH1 --- p.133 / Chapter 4.2.4.4 --- Putative downstream targets of FIH1 --- p.139 / Chapter 4.2.5 --- Identification of novel miR-31 targets by gene expression microarray --- p.139 / Chapter 4.3 --- Discussion --- p.145 / Chapter Chapter 5 --- MicroRNAs regulation on NPC stem-like properties --- p.154 / Chapter 5.1 --- Introduction --- p.154 / Chapter 5.2 --- Results --- p.156 / Chapter 5.2.1 --- MicroRNA expression profiles in NPC sphere-forming cells --- p.155 / Chapter 5.2.2 --- Ectopic expression of miR-183 family and miR-203 in NPC --- p.161 / Chapter 5.2.2.1 --- Sphere-forming ability of NPC cells --- p.161 / Chapter 5.2.2.2 --- Clone-forming ability of C666-1 --- p.161 / Chapter 5.2.3 --- Sphere-forming ability of NPC cells transfected with anti-miR-96 and anti-miR-183 --- p.164 / Chapter 5.2.4 --- Expression of cacner stem cell markers in NPC cells transfected with miR-96 and miR-183 --- p.164 / Chapter 5.3 --- Discussion --- p.167 / Chapter Chapter 6 --- General discussion --- p.170 / Reference --- p.177

The etiology of nasopharyngeal carcinoma (NPC) is based on intricate interactions among environmental factors, genetic susceptibility and Epstein-Barr virus (EBV) infection. Information concerning the role of EBV infection, particularly during the early stage of NPC development is poorly understood. Our laboratory has shown that stable infection of EBV could be achieved in immortalized epithelial cell lines which harbor genetic alterations and altered cell signaling pathway. In this study, these cell models were used to elucidate early events involved in EBV infection in premalignant nasopharyngeal epithelial cell models and their implications on development and progression of nasopharyngeal carcinoma. The response of EBV-infected cells to a stromal inflammatory cytokine, interleukin-6 (IL-6), was examined. EBV infection and long-term propagation of EBV-infected nasopharyngeal epithelial cells confer enhanced sensitivity to STAT3 activation induced by IL-6. IL-6-induced STAT3 activation reinforced their malignant properties in nasopharyngeal epithelial cells and may play a role in the development of nasopharyngeal carcinoma. Furthermore, constitutive STAT3 activation was demonstrated to facilitate malignant transformation of EBV-infected premalignant nasopharyngeal epithelial cells to cancer cells, suggesting that EBV infection and STAT3 activation might synergistically promote the development of NPC. This study also provides support for the existence of a positive feedback loop of IL-6/STAT3/LMP in NP460hTert-EBV cells, which enhanced STAT3 activation in EBV-infected cells. Elevated levels of IL-6Rα expression were observed in EBV-infected NP460hTert cells compared with uninfected cells and were largely responsible for the enhanced sensitivity of IL-6-induced STAT3 activation in these cells. High expression level of IL-6Rα could amplify IL-6 signaling in nasopharyngeal epithelial cells to promote growth proliferation in NP460hTert cells and increase the growth rate of xenografted NPC cells in immune-suppressed animals, suggesting that IL-6Rα overexpression may play a role of contributing to the development of nasopharyngeal carcinoma. The serum concentrations of both IL-6 and sIL-6R were also higher in NPC patients than healthy individuals and may have prognostic values to predict clinical outcome and disease progression in NPC patients. In conclusion, these data support the hypothesis that EBV infection under inflammatory environment may activate aberrant gene expressions and cell signaling to facilitate malignant transformation. The inflammatory cytokine, IL-6, may mediate the role of EBV infection in the development of NPC. / published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 5 uppsatser.

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / Pathology / Doctoral / Doctor of Philosophy