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Conserved sequences of BART and BHRF regions encoding viral microRNAs in Epstein-Barr virus-associated lymphoma.

ABSTRACT: Epstein-Barr virus (EBV) encodes at least 25 pri-microRNAs (miRNAs) in two regions of its DNA genome, BART and BHRF. B95-8, an EBV reference strain, has a deletion in the BART region. However, no information is available on the deletions or mutations in the BART and BHRF regions in clinical samples of EBV-associated lymphoma.Nine DNA fragments encoding miR-BARTs and two coding miR-BHRF1s were amplified by PCR from DNA samples extracted from 16 cases of EBV-associated lymphoma. All the PCR products were sequenced directly. DNA fragments encoding miR-BARTs and miR-BHRF1-1 were successfully amplified from all samples. An adenine-to-guanine mutation in the DNA fragment encoding miR-BART2-3p was detected in four of the 16 cases, and a cytosine-to-thymidine mutation in the DNA fragment encoding miR-BART11-3p was detected in one of the 16 samples. These mutations were not associated with any histological categories of lymphoma. In conclusion, mutations were rarely observed in the DNA encoding viral miRNAs in cases of lymphoma. This suggests that the DNA sequences of EBV-encoded miR-BARTs and miR-BHRF1-1 are conserved in EBV-associated lymphoma.

SUBMITTER: Sunagawa K

PROVIDER: S-EPMC5513352 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Conserved sequences of BART and BHRF regions encoding viral microRNAs in Epstein-Barr virus-associated lymphoma.

Sunagawa Keishin K Hishima Tsunekazu T Fukumoto Hitomi H Hasegawa Hideki H Katano Harutaka H

BMC research notes 20170714 1

<h4>Objective</h4>Epstein-Barr virus (EBV) encodes at least 25 pri-microRNAs (miRNAs) in two regions of its DNA genome, BART and BHRF. B95-8, an EBV reference strain, has a deletion in the BART region. However, no information is available on the deletions or mutations in the BART and BHRF regions in clinical samples of EBV-associated lymphoma.<h4>Results</h4>Nine DNA fragments encoding miR-BARTs and two coding miR-BHRF1s were amplified by PCR from DNA samples extracted from 16 cases of EBV-assoc ...[more]

PMID: 28705173

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Project description:UnlabelledEpstein-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 mutations into the putative NF-κB binding sites abolished activation of BART promoters by NF-κB. Binding of p50 subunits to NF-κB 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-κB activity in EBV-infected epithelial cells, while treatment of EBV-harboring NPC C666-1 cells with aspirin (acetylsalicylic acid [ASA]) and the IκB kinase inhibitor PS-1145 inhibited NF-κB activity, resulting in downregulation of BART expression. Expression of EBV LMP1 activates BART promoters, whereas an LMP1 mutant which cannot induce NF-κB activation does not activate BART promoters, further supporting the idea that expression of BARTs is regulated by NF-κB 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-κB 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.ImportanceNasopharyngeal 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-κB signaling. EBV LMP1 is a potent activator of NF-κB signaling, and we demonstrate that LMP1 can upregulate expression of BARTs through NF-κB signaling and that BART miRNAs are also able to downregulate LMP1 expression. It appears that aberrant NF-κB signaling and expression of BARTs form an autoregulatory loop for maintaining EBV latency in NPC cells. Further exploration of how targeting NF-κB signaling interrupts EBV latency in NPC cells may reveal new options for NPC treatment.

Project description:BACKGROUND:Burkitt lymphoma is an aggressive B-cell lymphoma presenting in three clinical forms: endemic, sporadic and immunodeficiency-associated. More than 90% of endemic Burkitt lymphoma carry latent Epstein-Barr virus, whereas only 20% of sporadic Burkitt lymphoma are associated with Epstein-Barr infection. Although the Epstein-Barr virus is highly related with the endemic form, how and whether the virus participates in its pathogenesis remains to be fully elucidated. In particular, the virus may impair cellular gene expression by its own encoded microRNAs. METHODS:Using microRNA profiling we compared Epstein-Barr-positive and Epstein-Barr-negative Burkitt lymphoma cases for both cellular and viral microRNAs. The array results were validated by qRT-PCR, and potential targets of viral microRNAs were then searched by bioinformatic predictions, and classified in functional categories, according to the Gene Ontology. Our findings were validated by in vitro functional studies and by immunohistochemistry on a larger series of cases. RESULTS:We showed that a few cellular microRNAs are differentially expressed between Epstein-Barr-positive and Epstein-Barr-negative Burkitt lymphoma cases, and identified a subset of viral microRNAs expressed in Epstein-Barr-positive Burkitt lymphomas. Of these, we characterized the effects of viral BART6-3p on regulation of cellular genes. In particular, we analyzed the IL-6 receptor genes (IL-6R? and IL-6ST), PTEN and WT1 expression for their possible relevance to Burkitt lymphoma. By means of immunohistochemistry, we observed a down-regulation of the IL-6 receptor and PTEN specifically in Epstein-Barr-positive Burkitt lymphoma cases, which may result in the impairment of key cellular pathways and may contribute to malignant transformation. On the contrary, no differences were observed between Epstein-Barr-positive and Epstein-Barr-negative Burkitt lymphoma cases for WT1 expression. CONCLUSIONS:Our preliminary results point at an active role for the Epstein-Barr virus in Burkitt lymphomagenesis and suggest new possible mechanisms used by the virus in determining dysregulation of the host cell physiology.

Dataset Information

Conserved sequences of BART and BHRF regions encoding viral microRNAs in Epstein-Barr virus-associated lymphoma.

Publications

Conserved sequences of BART and BHRF regions encoding viral microRNAs in Epstein-Barr virus-associated lymphoma.

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