Project description:Here, we report a systematic effort to identify pro-apoptotic mRNA targets for the EBV miR-BART miRNAs. We demonstrate that at least five of the 22 miR-BART pre-miRNAs have anti-apoptotic activity and we identify seven pro-apoptotic cellular mRNA targets, six of them novel, that contribute significantly to the observed anti-apoptotic phenotype. Together, these data represent a substantial increase in our understanding of the role of the miR-BART miRNAs in promoting EBV infection and latency. Use of deep sequencing to identify all the miRNAs expressed in C666 cells and PAR-CLIP to comprehensively profile EBV miRNA targets in C666 cells.
Project description:Here, we report a systematic effort to identify pro-apoptotic mRNA targets for the EBV miR-BART miRNAs. We demonstrate that at least five of the 22 miR-BART pre-miRNAs have anti-apoptotic activity and we identify seven pro-apoptotic cellular mRNA targets, six of them novel, that contribute significantly to the observed anti-apoptotic phenotype. Together, these data represent a substantial increase in our understanding of the role of the miR-BART miRNAs in promoting EBV infection and latency.
Project description:The Epstein-Barr virus (EBV) encodes its own microRNAs (miRNAs); however, their biological roles remain elusive. The commonly used EBV B95-8 strain lacks a 12 kb genomic region, known as BamHI A Rightward Transcripts (BART) locus, which encodes a number of viral miRNAs (BART miRNAs). These miRNAs are expressed abundantly in EBV-positive epithelial malignancies, suggesting that the 12 kb region somehow contributes to EBV-mediated epithelial carcinogenesis. Bacterial artificial chromosome (BAC) technology was used to generate an EBV B95-8 strain in which the 12 kb region was fully restored at its native locus (BART-restored virus). HEK293 and AdAH cells infected with either the parental BART-deleted virus or the BART-restored virus were established. The gene expression profiles of these cells were examined to identify cellular target genes of BART miRNAs.
Project description:The Epstein-Barr virus (EBV) encodes its own microRNAs (miRNAs); however, their biological roles remain elusive. The commonly used EBV B95-8 strain lacks a 12 kb genomic region, known as BamHI A Rightward Transcripts (BART) locus, which encodes a number of viral miRNAs (BART miRNAs). These miRNAs are expressed abundantly in EBV-positive epithelial malignancies, suggesting that the 12 kb region somehow contributes to EBV-mediated epithelial carcinogenesis. Bacterial artificial chromosome (BAC) technology was used to generate an EBV B95-8 strain in which the 12 kb region was fully restored at its native locus (BART-restored virus). HEK293 and AdAH cells infected with either the parental BART-deleted virus or the BART-restored virus were established. The gene expression profiles of these cells were examined to identify cellular target genes of BART miRNAs. Total RNAs of 2 independent v-miRNA-positive HEK293 (or AdAH) cells and 2 independent v-miRNA-negative cells HEK293 (or AdAH) cells were processed for Microarray analysis using 3D-Gene human Oligo chip 25k (Toray, Tokyo, Japan).
Project description:Epstein-Barr virus (EBV) is highly successful virus infecting the majority of the human population worldwide. During the latent infection period, there are only a few of EBV-encoded proteins can be detected, whereas EBV-encoded non-coding RNAs are highly activated, especially microRNAs. Recent studies found that those BART microRNAs not only disturb EBV genes expression, but also interfere many host genes expression, thus modulating cellular proliferation and immune regulation. In the present study, we investigate the effect of EBV BART6-3p on gene expression profile of the human PBMCs.
Project description:Cellular targets for most of EBV miRNAs are not known. In our study, we aimed at identifying genes that are regulated by individual EBV mature miRNA, particularly BART 18-5p We used microarrays to explore the global gene expression (particularly the downregulated genes) targeted by EBV miRNA mimics. Burkitt's lymphoma cell lines, BL2 and BJAB, were transfected with miRNA mimic control (MC) and EBV miRNA mimics BART 18-5p, 10 and 14* for 24 hour prior to RNA extraction and hybridization on Affymetrix human HGU133 plus 2.0 microarrays.
Project description:Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus associated with nasopharyngeal carcinoma (NPC). EBV encodes two groups of microRNAs (miRNAs) which are divided into BamHI fragment H rightward open reading frame 1 (BHRF1) and BamHI-A rightward transcripts (BART) microRNAs. EBV miR-BARTs have been found to be involved in the development and progression of NPC. However, the role of EBV-miR-BARTs in NPC progression remains illusive. This study aimed to investigate the role of EBV-miR-BARTs in NPC and explore the underlying mechanisms.
Project description:Transcriptional profiling of Burkitt lymphoma cells engineered to inducibly express dominant negative EBNA1 (dnEBNA1), which forces the loss of Epstein Barr virus (EBV). Profiles are made of cells in which all of EBV is lost (dnEBNA1 on) or all of EBV except the BART miRNAs is lost (dnEBNA1 on, BART miRNAs ectopically expressed). This is a 2-condition experiment: uncomplemented cells (EBV evicted with dnEBNA1), and BART-complemented cells (EBV evicted with dnEBNA1, BART miRNAs expressed ectopically). There are 3 biological replicates for each condition.
Project description:MicroRNAs (miRNAs) represent a conserved class of small non-coding RNAs that are found in all higher eukaryotes as well as some DNA viruses. MiRNAs are 20-25 nucleotides (nt) in length and have important regulatory functions in biological processes such as embryonic development, cell differentiation, hormone secretion or metabolism. Furthermore, miRNAs have been implicated in the pathology of various diseases including cancer. MiRNA expression profiles not only classify different types of cancer but also may even help to characterize distinct tumor stages, therefore constituting a valuable tool for prognosis. Here we report the miRNA profile of Epstein-Barr Virus (EBV)-positive nasopharyngeal carcinoma (NPC) tissue samples characterized by cloning and sequencing. We find that all EBV miRNAs from the BART region are expressed in NPC tissues whereas ebv-miRNAs from the BHRF1 region are not found. Moreover, we identify two novel EBV miRNA genes originating from the BART region that have not been found in other tissues or cell lines before. We also identify three new human miRNAs, which might be specific for nasopharyngeal tissues. We further show that a number of different cellular miRNAs are upor down-regulated in NPC tissues compared to control tissue including miR-15a and miR-16. We find that the tumor suppressor BRCA-1 is a target of miR-15a as well as miR-16 suggesting a miRNA role in NPC pathogenesis. 2 pairs of NPC and control tissues from 2 patients (4 samples in total) were examined.
Project description:The human herpesvirus Epstein-Barr virus establishes persistent infections in the majority of the human population. Its main target cells are B lymphocytes and epithelial cells lining the oropharynx. Although primary infection usually occurs asymptomatically, EBV can cause mononucleosis. Moreover, EBV is strongly associated with multiple malignancies, including nasopharyngeal carcinoma, Burkitt Lymphoma, Hodgkin’s Lymphoma and post-transplant lymphoproliferative disease. In these EBV+ tumour tissues, the virus is latent and expresses only few proteins, yet up to ~40 miRNAs. Past research has indicated a possible causative link between these miRNAs and tumour development. However, the functions of these miRNAs remain poorly characterized. We aimed to explore the targetome of the EBV BART miRNAs by expressing these in a nasopharyngeal carcinoma cell line and performing whole genome gene expression profiling. Indeed, we identified numerous differentially expressed genes between miRNA-positive and -negative cells. By applying 3'UTR reporter assays, we validated eight genes as direct BART miRNA targets and identified the responsible miRNA(s). We found evidence for co-regulation of certain target genes by multiple miRNAs. Amongst these newly identified target genes was ATG16L1, a gene essential for autophagy. ATG16L1 protein levels were repressed by miR-BART18 in EBV+ Burkitt Lymphoma cells.