published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Clinical Oncology / Master / Master of Philosophy

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / Surgery / Master / Master of Philosophy

Genes identified by SAGE may serve as potential prognostic marker or therapeutic target. 14-3-3 sigma is a putative tumor suppressor and can be induced in response to DNA damage following irradiation, leading to cell cycle arrest in G2/M in human cancer cells. Our SAGE results revealed that 14-3-3 sigma expression is significantly downregulated in C666-1 cells. The study of 72 primary NPCs showed that an increased expression of 14-3-3 sigma was associated with a poorer clinical outcome in terms of shorter overall survival (OS; p=0.0297) and shorter disease free survival (DFS; p=0.042) using univariate analysis. Hence, 14-3-3 sigma may be used as an independent prognostic marker for NPC patients. / In conclusion, a NPC transcription profile has been successfully generated and several candidate NPC-associated genes have been identified by Serial Analysis of Gene Expression (SAGE) and NPC transcriptome map. These novel findings lead to better understanding of the molecular basis of NPC development and provide a foundation for discovery of new therapeutic strategies. / Nasopharyngeal carcinoma (NPC) is one of the most prevalent cancers among Southern Chinese. To better understand the genetic basis of this disease, Serial Analysis of Gene Expression (SAGE) was performed to investigate the transcriptional profiles of an EBV-positive NPC cell line (C666-1) and a normal NP outgrowth (NP4). A total of 102,059 SAGE tags were extracted in both libraries and 250 genes with 10-fold or more differential expression were found in NPC cells compared to normal NP cells. Eleven differentially expressed genes identified by SAGE were selected for confirmation using real time RT-PCR. The transcripts for 5 of the 11 genes, CD 74, Transcriptional intermediary factor 1, Ferritin 1, Claudin 4, and fatty acid synthase were overexpressed in NPC cells. Conversely, the remaining transcripts including Keratin 17, Keratin 5, S100 calcium-binding A2, Cystatin A, 14-3-3 sigma and Caveolin 1 were underexpressed in NPC cells. The aberrant expression of CD74, Claudin 4, Fatty acid synthase, 14-3-3 sigma, Caveolin 1 were further validated by immunohistochemistry on 20 NPC patients. / On the other hand, fatty acid synthase (FASN), a key enzyme for de novo lipogenesis, is a putative therapeutic target in treating NPC. Immunohistochemical studies showed upregulation of FASN in 20.8% (15/72) of the NPC cases compared with the adjacent normal NP epithelium. In addition, FASN expression also had prognostic significance in predicting the outcome of patients after radiotherapy, as high levels of FASN expression were associated with worse overall survival (OS, p=0.032) and disease free survival (DFS, p=0.002) in NPC patients. FASN inhibitors, such as C75 which inhibit cell growth via cell cycle arrest in G2/M phase, are potential chemotherapeutic agents in treating NPC. / The genome-wide quantitative analysis of gene expression by SAGE with matched chromosomal positions enables the construction of a transcriptome map of NPC. A total of 8 and 29 overexpressed and underexpressed gene clusters were observed, respectively. Some novel regions that have never been illustrated in previous reports such as amplification regions at 2p11.2-p25.1, 2q33-q37, 9q22-q34, 17p11.2-p13.2 and deletion regions at 1p12-p31.2, 1q25-q42.12, 2q21.3-q33, 8p21.1-p22, 9q33-q34.3, 10q23.3-q26.3, 12p13, 16p13, 17q23.2-q25, 19p13, 19q12-q13.2, 20p11-p13, 22q13, Xp11.2-p11.4, and Xq26-q28 were also identified. A candidate tumor suppressor gene named MEG3 has been found within an underexpressed region at 14q32.2 in the NPC transcriptome map. Our FISH analysis revealed that chromosome loss at 14q32 is associated with hypermethylation of MEG3 promoter region in 9/13 (75%) of NPC patients. Loss of imprinting is the major mechanism that governs the MEG3 expression. Moreover, transient transfection of one of the MEG3 isoforms (accession no. AF119863) could obviously inhibit cell colony formation of NPC cells. Taken together, MEG3 gene on chromosome 14q32.2 might act as a tumor suppressor in NPC. / Chan, Yat Yee. / "March 2008." / Adviser: Lo Kwok Wai. / Source: Dissertation Abstracts International, Volume: 70-03, Section: B, page: 1605. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (p. 196-225). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Pathology / Master / Master of Medical Sciences

Epstein-Barr virus (EBV) is one of the most successful human pathogens in the world and establishes a lifelong persistent infection in 95% of adult population worldwide. It is associated with a number of malignancies including Burkett’s lymphoma, Hodgkin’s lymphoma, nasopharyngeal carcinoma(NPC) and gastric carcinoma. EBV was the first virus reported to produce microRNAs (miRNAs) and it encodes 44 mature miRNAs from 2 viral transcripts, BART and BHRF1. The BART transcript is abundantly expressed in all latently infected cells, particularly in epithelial cells. The BART miRNAs (miR-BARTs) were shown to be involved in apoptosis inhibition, immune evasion, metastasis, viral and cellular transcripts regulation. The high expression profile and the diverse functions of miR-BARTs suggest that they may play a critical role in the development of EBV-associated NPC. In order to understand the importance of miR-BARTs in NPC development, in this thesis, I conducted a study on the miR-BARTs function in nasopharyngeal carcinogenesis. In the first part, I characterized the cellular target and function of an abundantly expressed miR-BART in NPC. In the second part, I established a novel recombinant EBV construction system for genetic studies of miR-BARTs in nasopharyngeal epithelial (NP) cells. In the first part of my study, I characterized the cellular target and function of miR-BART3* in NPC. As predicted by bioinformatics, tumor suppressor protein DICE1 was a cellular target of miR-BART3*. The specific targeting between miR-BART3* and DICE1 3’UTR was validated by luciferase assays and the downregulation of both endogenous DICE1 protein and mRNA was observed in EBV+epithelial cells and miR-BART3* expressing cells. In addition, restoration of DICE1 protein expression by inhibition of miR-BART3* was also demonstrated in EBV+epithelial cells. Moreover, miR-BART3* was shown to promote cell proliferation via suppression of DICE1. Analysis of22 human nasopharyngeal(NP)biopsy samples demonstrated the inverse correlation between miR-BART3* and DICE1 expression. Taken together, miR-BART3* downregulates the tumor suppressor DICE1 protein to promote cell proliferation and transformation in NPC. Besides the candidate approach, genetic studies can provide a systematic view of the functions of all miR-BARTsand shed light on the importance of miR-BARTs in NPC under a more physiological condition. At present, bacterial artificial chromosome (BAC) technology is commonly used for recombinant EBV construction. However, the intrinsic disadvantages of BAC prevent its use in NP epithelial cells. Therefore in the second part of my study, I established a novel CRISPR/Cas9-mediated recombinant EBV construction system and constructed a miR-BART deleted recombinant EBV. The CRISPR/Cas9 system was demonstrated to be effective in EBV genome editing and Akata cells were infected by the recovered recombinant mutant virus. Infected Akata cells served as the source for NP cell infection through co-culture. The new CRISPR/Cas9 system have many advantages over the conventional EBV BAC method. My work reported in this thesis not only illustrated the importance of miR-BARTs in NPC, but also provide a new technology platform for further study of miR-BARTs in NP epithelial cells. (An / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy