Oncogenic role of epstein-barr virus Lnc-BARTs in nasopharyngeal carcinoma

Abstract

Epstein-Barr virus (EBV) is a ubiquitous human virus and one of the key etiological factors associated with oncogenic progress of nasopharyngeal carcinoma (NPC) . In NPC cells, EBV expresses very few viral proteins in its latent state, which is believed to facilitate evasion of immune surveillance, but consistently high levels of long non-coding RNAs, (lnc-BARTs), which are derived from multiple exons in the BamHI-A rightward transcript (BART) region of the EBV genome. However, details of the biological functions of these EBV-encoded long non-coding RNAs remain elusive. My study aims to provide experimental evidence to show that lnc-BARTs function as regulatory RNAs in modulating core network activity through epigenetic to maintain EBV latency in NPC cells. My study first identified that EBV lnc-BARTs localize in nuclear speckles, highly dynamic subdomains of the nucleus enriched with transcription, elongation and splicing factors. To better understand the role of lnc-BARTs in NPC, I used CRISPR-Cas9 technology to achieve stable-knockdown of lnc-BARTs in the EBV- harbouring C666-1 NPC cell line. I found that expression of the EBV latent genes EBNA1 and LMP1 is also down-regulated following the knockdown of lnc-BARTs. Importantly, I also discovered that knockdown of lnc-BARTs resulted in downregulation of the oncogenes MYC and BCL2. In addition, ATAC sequencing showed that the depletion of lnc-BARTs induced apoptosis and decreased accessibility of open chromatin in NPC cells. I found that lnc-BARTs physically interact with a complex consisting of several transcription factors, including BRD4 and KDM1B. I further showed that KDM1B increased the levels of H3K4me1/2 in NPC cells. Notably, RNA FISH analysis indicated that lnc-BARTs co-localize with BRD4 and positive transcription elongation factor (P-TEFb) in nuclear speckles; these complexes can be disrupted by treatment with JQ1, a competitive inhibitor of BRD4. Using RIP and RNA pulldown assays, my study demonstrated that lnc-BARTs bind directly to BRD4 and act as a scaffold to mediate an association between BRD4 and the splicing factor SC35. Moreover, EBV lnc-BARTs are also involved in the P-TEFb regulatory machinery via their association with BRD4. BRD4-ChIP analysis further demonstrated that lnc-BARTs recruit the BRD4-mediated epigenetic modulator complex to the promoter regions of MYC and BCL2. In summary, my thesis provides the first evidence of the mechanisms underlying the critical role of lnc-BARTs in maintaining EBV latency and driving tumorigenesis in NPC. EBV lnc-BARTs modulate the expression of genes associated with tumor apoptosis during EBV latency through functional interaction with BRD4- and KDM1B-associated regulatory machinery in NPC cells.