Oncogenic role of Epstein-Barr virus lnc-BARTs in EBV associated tumors

Abstract

<p>While majority of humans infected with Epstein-Barr virus (EBV) is asymptomatic for whole life, EBV is found to associate with several human cancers, including Burkitt’s lymphoma, Hodgkin’s diseases, post-transplant lymphoproliferative disorder (PTLD), nasopharyngeal carcinoma (NPC) and gastric carcinoma. Among these EBV associated tumors, EBV genome is found in 100% of NPC tumor cells. EBV establishes a latent infection in NPC cells, expressing few viral proteins, but elevated levels of non-coding RNAs transcribed from the BamHI-A region of the EBV genome, designated BamHI-A rightward transcripts (BARTs). The family of EBV BART RNAs comprises BART-microRNAs (miRNAs) and long non-coding RNAs (lnc-BARTs). While versatile functions of BART miRNAs have been revealed, little is known about the role of EBV lnc-BARTs. Here, we provided evidence to show that BARTs mRNA which derived from multiple exons of BART function as regulatory long non-coding RNA, namely lnc-BARTs, in modulating the core network for maintaining EBV latency and promoting NPC oncogenesis through epigenetic mechanisms. Our results showed that the apoptosis rate of NPC cell lines with knockdown of EBV lnc-BARTs was significantly increased, suggesting that lnc-BARTs have an anti-apoptotic effect in EBV associated tumors. MS analysis was performed to identify host factors which interact with EBV lnc-BARTs in NPC cells. Lnc-BARTs were shown to directly interact with several transcriptional regulators, including BRD4 and SC35. Notably, RNA-FISH assays demonstrated that lnc-BARTs co-localize with BRD4 and SC35 complexes in nuclear speckles, with these complexes being disrupted by treatment with JQ1, a BRD4 competitive inhibitor. RNA immunoprecipitation and RNA pulldown assays confirmed that lnc-BARTs bind to BRD4. ATAC sequencing analysis and transcriptome analysis further demonstrated that knockdown of BARTs in the EBV-harboring NPC cell line, C666-1, resulted in globally reduced chromatin accessibility downregulation of oncogenes like MYC and BCL2. In addition, ChIP sequencing analysis revealed that lnc-BARTs interfere PolII phosphorylation on promoters of genes. Our findings suggest that EBV lnc-BARTs are involved in epigenetic modulation of host gene expression through functional interaction with elongation regulatory machinery to maintain EBV latency and drive tumorigenesis in NPC.<br></p>