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This dissertation, Functional Study of the EBV-encoded RNAs (EBERs) in Nasopharyngeal Epithelial Cells by Hing-lok, Wong, 黃慶樂, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled Functional Study of the EBV-Encoded RNAs (EBERs) in Nasopharyngeal Epithelial Cells Submitted by Hing Lok Wong for the degree of Doctor of Philosophy at The University of Hong Kong in July 2005 Infection of Epstein-Barr virus is associated with the development of malignancy including Burkitt's lymphoma, Hodgkin's disease and nasopharyngeal carcinoma. The EBV-encoded small RNAs (EBERs) are non-coding RNAs expressed at high levels in EBV-infected cells. The function role of EBERs in nasopharyngeal carcinoma is unclear. Studies of EBERs in Burkitt's lymphoma (BL) suggest that EBER may be involved in maintaining the malignant phenotypes. EBV infection is believed to play an important etiological role in nasopharyngeal carcinoma but there is no relevant cell model to delineate the role of EBV and its encoded gene products in the development of nasopharyngeal carcinoma. Recently, our laboratory has established an immortalized cell system (NP69) of nasopharyngeal epithelial cell origin that bears resemblances to premalignant nasopharyngeal epithelial cells in harboring multiple genetic alterations without attaining malignant phenotypes. In this study, the NP69 cell model was used to examine the functions of EBERs. High level expression of EBERs in NP69 cells was achieved by stable transfection of an EBER-expressing plasmid (EKS10). The growth rate of EBER-expressing NP69 was slightly enhanced, with the down-regulation of the cell INK4a Cip1 cycle regulators, p16 and p21, compared to control NP69 cells transfected with an empty vector. EBER expression however, did not result in tumorigenic transformation of NP69 cells. EBER expression in NP69 cells could down-regulate phosphorylation of PKR suggesting suppression of PKR activation (a host defense mechanism to viral infection and cellular stress, resulting in inhibition of protein synthesis and apoptosis). dsRNA is commonly associated with viral infection and EBERs have structural similarity to dsRNA and were shown previously to bind and inactivate PKR activity. The ability of EBER to interfere with PKR signaling in NP69 cells and the biological consequences were examined in this study. PKR activation in cells were achieved by treatment with poly(I)poly(C) [poly(I: C)], a dsRNA analogue. The results showed that EBER expression in NP69 cells could effectively suppress PKR phosphorylation and confer resistance to poly(I: C)-induced apoptosis. The potential pathways involved in resistance to poly(I: C)-induced apoptosis by EBERs was investigated by examining the proteins commonly involved in apoptosis (e.g. Bcl-2, Bax, caspase-3 and PARP) by Western blotting and gene expression profile using cDNA microarray analysis. Notably, the basal level of Bcl-2 expression was higher in the EBER-expressing cells compared to control cells. In addition, genes commonly activated by interferon were seen to be suppressed in EBER-expressing cells. The suppression of expression of interferon-responsive genes e.g. IL1A, IL6, OAS1, MX1, STAT1, IFIT1, IFI35, IRF-7 and CXCL1, were confirmed by real-time quantitative PCR. Phosphorylation of STAT1 is a common cellular signaling pathway activated by interferon. Suppression of STAT1 phosphorylation induced by interferon treatment was observed in EBER-expressing NP69 cells confirming a role of EBER in resisting the action of interferon. In summary, EBER expression

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