Roles of Epstein-Barr virus-encoded miR-BART microRNAs in viral infection of nasopharyngeal epithelial cells

Abstract

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