Oncogenic Role Of Epstein-Barr Virus Lnc-Barts In Nasopharyngeal Carcinoma Cells

Epstein-Barr virus (EBV) is associated with several human cancers, including Burkitt9s lymphoma, Hodgkin9s 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 BART RNAs comprises BART-microRNAs (miRNAs) and long non-coding RNAs (lnc-BARTs). While versatile functions of BART miRNAs continue to be revealed, little is known about EBV lnc-BARTs. Here, we provide evidence to show that lnc-BARTs derived from multiple exons of BART RNA function as nuclear long non-coding RNAs in modulating host gene expression in NPC cells. We found that lnc-BARTs upregulate transcription of SEPT9, matrix metalloproteinase (MMP) genes and IKZF3 (Aiolos) and accelerate N-cadherin cleavage. As a member of the Septin family, SEPT9 (cytoskeletal interacting protein, Septin 9) is involved in cytokinesis and cell cycle control. Aiolos can negatively regulate expression of p66Shc, which is associated with the oxidative stress response and apoptosis, and our data also showed that knockout of IKZF3 in NPC cells enhanced transcription of p66Shc protein. Our results indicated that the apoptosis rate of NPC cell lines with knockdown of lnc-BARTs was significantly increased, suggesting that lnc-BARTs have an anti-apoptotic effect in EBV associated tumors. To understand the molecular basis of gene regulation by EBV BART-lncRNAs, ChIRP-MS analysis was performed: lnc-BARTs were shown to directly interact with several transcriptional regulators, including BRD4 and KDM1B. Notably, RNA FISH assays demonstrated that lnc-BARTs co-localize with BRD4 and P-TEFb 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 directly to BRD4. In addition, transcriptome analysis revealed that knockdown of BARTs in the EBV-harboring NPC cell line, C666-1, resulted in downregulation of MYC and BCL2, genes downstream of BRD4. BRD4-ChIP and ATAC sequencing analysis further demonstrated that knockdown of lnc-BARTs reduced both BRD4-mediated epigenetic modulation of host gene expression and chromatin accessibility. We suggest that EBV lnc-BARTs are involved in epigenetic modulation of host gene expression through functional interaction with BRD4 regulatory machinery to maintain EBV latency in NPC cells, and that lnc-BARTs modulate host gene expression to drive tumorigenesis in NPC. Citation Format: Jiayan Liu, Bobo Wing-Yee Mok, Songtao He, Honglin Chen. Oncogenic role of epstein-barr virus lnc-BARTs in nasopharyngeal carcinoma cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 272.