Dataset Information

NF-?B Signaling Regulates Expression of Epstein-Barr Virus BART MicroRNAs and Long Noncoding RNAs in Nasopharyngeal Carcinoma.

ABSTRACT: UNLABELLED:Epstein-Barr virus (EBV) expresses few viral proteins in nasopharyngeal carcinoma (NPC) but high levels of BamHI-A rightward transcripts (BARTs), which include long noncoding RNAs (lncRNAs) and BART microRNAs (miRNAs). It is hypothesized that the mechanism for regulation of BARTs may relate to EBV pathogenesis in NPC. We showed that nuclear factor-?B (NF-?B) activates the BART promoters and modulates the expression of BARTs in EBV-infected NPC cells but that introduction of mutations into the putative NF-?B binding sites abolished activation of BART promoters by NF-?B. Binding of p50 subunits to NF-?B sites in the BART promoters was confirmed in electrophoretic mobility shift assays (EMSA) and further demonstrated in vivo using chromatin immunoprecipitation (ChIP) analysis. Expression of BART miRNAs and lncRNAs correlated with NF-?B activity in EBV-infected epithelial cells, while treatment of EBV-harboring NPC C666-1 cells with aspirin (acetylsalicylic acid [ASA]) and the I?B kinase inhibitor PS-1145 inhibited NF-?B activity, resulting in downregulation of BART expression. Expression of EBV LMP1 activates BART promoters, whereas an LMP1 mutant which cannot induce NF-?B activation does not activate BART promoters, further supporting the idea that expression of BARTs is regulated by NF-?B signaling. Expression of LMP1 is tightly regulated in NPC cells, and this study confirmed that miR-BART5-5p downregulates LMP1 expression, suggesting a feedback loop between BART miRNA and LMP1-mediated NF-?B activation in the NPC setting. These findings provide new insights into the mechanism underlying the deregulation of BARTs in NPC and identify a regulatory loop through which BARTs support EBV latency in NPC. IMPORTANCE:Nasopharyngeal carcinoma (NPC) cells are ubiquitously infected with Epstein-Barr virus (EBV). Notably, EBV expresses very few viral proteins in NPC cells, presumably to avoid triggering an immune response, but high levels of EBV BART miRNAs and lncRNAs which exhibit complex functions associated with EBV pathogenesis. The mechanism for regulation of BARTs is critical for understanding NPC oncogenesis. This study provides multiple lines of evidence to show that expression of BARTs is subject to regulation by NF-?B signaling. EBV LMP1 is a potent activator of NF-?B signaling, and we demonstrate that LMP1 can upregulate expression of BARTs through NF-?B signaling and that BART miRNAs are also able to downregulate LMP1 expression. It appears that aberrant NF-?B signaling and expression of BARTs form an autoregulatory loop for maintaining EBV latency in NPC cells. Further exploration of how targeting NF-?B signaling interrupts EBV latency in NPC cells may reveal new options for NPC treatment.

SUBMITTER: Verhoeven RJ

PROVIDER: S-EPMC4936125 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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NF-κB Signaling Regulates Expression of Epstein-Barr Virus BART MicroRNAs and Long Noncoding RNAs in Nasopharyngeal Carcinoma.

Verhoeven Rob J A RJ Tong Shuang S Zhang Gaohong G Zong Jingfeng J Chen Yixin Y Jin Dong-Yan DY Chen Mei-Ru MR Pan Jianji J Chen Honglin H

Journal of virology 20160624 14

<h4>Unlabelled</h4>Epstein-Barr virus (EBV) expresses few viral proteins in nasopharyngeal carcinoma (NPC) but high levels of BamHI-A rightward transcripts (BARTs), which include long noncoding RNAs (lncRNAs) and BART microRNAs (miRNAs). It is hypothesized that the mechanism for regulation of BARTs may relate to EBV pathogenesis in NPC. We showed that nuclear factor-κB (NF-κB) activates the BART promoters and modulates the expression of BARTs in EBV-infected NPC cells but that introduction of mu ...[more]

PMID: 27147748

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Project description:This study assessed the effects of Epstein-Barr virus (EBV) and one form of virally encoded BART long noncoding RNAs (lncRNAs) on cellular expression in epithelial cells grown in vitro and as tumors in vivo determined by high-throughput RNA sequencing of mRNA and small RNAs. Hierarchical clustering based on gene expression distinguished the cell lines from the tumors and distinguished the EBV-positive tumors and the BART tumors from the EBV-negative tumors. EBV and BART expression also induced specific expression changes in cellular microRNAs (miRs) and lncRNAs. Multiple known and predicted targets of the viral miRs, the induced cellular miRs, and lncRNAs were identified in the altered gene set. The changes in expression in vivo indicated that the suppression of growth pathways in vivo reflects increased expression of cellular miRs in all tumors. In the EBV and BART tumors, many of the targets of the induced miRs were not changed and the seed sequences of the nonfunctional miRs were found to have homologous regions within the BART lncRNA. The inhibition of these miR effects on known targets suggests that these induced miRs have reduced function due to sponging by the BART lncRNA. This composite analysis identified the effects of EBV on cellular miRs and lncRNAs with a functional readout through identification of the simultaneous effects on gene expression. Major shifts in gene expression in vivo are likely mediated by effects on cellular noncoding RNAs. Additionally, a predicted property of the BART lncRNA is to functionally inhibit the induced cellular miRs. IMPORTANCE This study identified the total effects of EBV and a viral long noncoding RNA (BART lncRNA) on cellular RNA expression when grown as cells in culture and when grown as tumors in immunodeficient mice. The effects on cellular mRNA expression, lncRNA expression, and cellular and viral miR expression were determined using next-generation sequencing (NGS) and bioinformatics functional analysis. Many cellular growth pathways that are activated during growth in culture are decreased during growth as tumors. This study shows that these changes in expression are accompanied by induction of cellular-growth-inhibitory miRs. However, in the EBV tumors and in tumors expressing the BART lncRNA, many of the known targets of the inhibitory miRs are not affected. Regions of strong homology to the seed sequences of these miRs were identified in the BART lncRNA. These findings suggest that the BART lncRNA functions as a sponge for growth-inhibitory miRs.

Project description:Latent membrane protein 1 (LMP1) is an Epstein-Barr virus (EBV)-encoded oncoprotein that is packaged into small extracellular vesicles (EVs) called exosomes. Trafficking of LMP1 into multivesicular bodies (MVBs) alters the content and function of exosomes. LMP1-modified exosomes enhance the growth, migration, and invasion of malignant cells, demonstrating the capacity to manipulate the tumor microenvironment and enhance the progression of EBV-associated cancers. Despite the growing evidence surrounding the significance of LMP1-modified exosomes in cancer, very little is understood about the mechanisms that orchestrate LMP1 incorporation into these vesicles. Recently, LMP1 was shown to be copurified with CD63, a conserved tetraspanin protein enriched in late endosomal and lysosomal compartments. Here, we demonstrate the importance of CD63 presence for exosomal packaging of LMP1. Nanoparticle tracking analysis and gradient purification revealed an increase in extracellular vesicle secretion and exosomal proteins following LMP1 expression. Immunoisolation of CD63-positive exosomes exhibited accumulation of LMP1 in this vesicle population. Functionally, CRISPR/Cas9 knockout of CD63 resulted in a reduction of LMP1-induced particle secretion. Furthermore, LMP1 packaging was severely impaired in CD63 knockout cells, concomitant with a disruption in the perinuclear localization of LMP1. Importantly, LMP1 trafficking to lipid rafts and activation of NF-κB and PI3K/Akt pathways remained intact following CD63 knockout, while mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and noncanonical NF-κB activation were observed to be increased. These results suggest that CD63 is a critical player in LMP1 exosomal trafficking and LMP1-mediated enhancement of exosome production and may play further roles in limiting downstream LMP1 signaling.IMPORTANCE EBV is a ubiquitous gamma herpesvirus linked to malignancies such as nasopharyngeal carcinoma, Burkitt's lymphoma, and Hodgkin's lymphoma. In the context of cancer, EBV hijacks the exosomal pathway to modulate cell-to-cell signaling by secreting viral components such as an oncoprotein, LMP1, into host cell membrane-bound EVs. Trafficking of LMP1 into exosomes is associated with increased oncogenicity of these secreted vesicles. However, we have only a limited understanding of the mechanisms surrounding exosomal cargo packaging, including viral proteins. Here, we describe a role of LMP1 in EV production that requires CD63 and provide an extensive demonstration of CD63-mediated exosomal LMP1 release that is distinct from lipid raft trafficking. Finally, we present further evidence of the role of CD63 in limiting LMP1-induced noncanonical NF-κB and ERK activation. Our findings have implications for future investigations of physiological and pathological mechanisms of exosome biogenesis, protein trafficking, and signal transduction, especially in viral-associated tumorigenesis.

Dataset Information

NF-?B Signaling Regulates Expression of Epstein-Barr Virus BART MicroRNAs and Long Noncoding RNAs in Nasopharyngeal Carcinoma.

Publications

NF-κB Signaling Regulates Expression of Epstein-Barr Virus BART MicroRNAs and Long Noncoding RNAs in Nasopharyngeal Carcinoma.

Similar Datasets

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