Expression data from Burkitt Lymphoma cell lines followed by expression or inhibition of ebv-BART6-3p microRNA
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ABSTRACT: Epstein-Barr virus is associated with several human malignancies, including Burkitt Lymnphoma. The virus encodes more than 40 microRNAs, which participate in its possible pathogenetic role. We used microarrays to study the effect of the expression of an Epstein-Barr virus-encoded microRNA (ebv-BART6-3p) on the global gene expression profile of Burkitt Lymphoma cell lines. In order to determine the BART6-3p targets, EBV-negative Akata 2A8 or EBV-positive Akata cells were transiently transfected (in duplicates) with synthetic BART6-3p mimic or inhibitor (100 nM; Custom synthesized by Dharmacon- Thermo Scientific, Germany), respectively. For each treatment, a further transfection with corresponding negative control was performed as well (10 nM; I-300145-01, Dharmacon- Thermo Scientific, Germany). The transfection of 5,000,000 cells per treatment was performed by Amaxa nucleofector apparatus (Amaxa, Cologne-Germany), using the program G23 and the transfection solution V according to the manufacturerâs instructions. Transfection efficiency was assessed by means of a further treatment (2µg of pmaxGFP) and detection of both fluorescence and cell viability by flow cytometry. Twenty four hours post-transfection, cells were harvested and RNA was extracted using Trizol, and transfection efficiency was further confirmed by evaluating the expression level of BART6-3p using q-PCR by means of Taqman probes, employing RNU43 as housekeeping miRNA (Applied Biosystems, Cologne, Germany). RNA was further hybridized to HuGene-2.0-st array (Affymetrix, Santa Clara, CA) according to the manufacturer's instructions.
Project description:Transition of Akata Burkitt lymphoma (BL) cells from a malignant to nonmalignant phenotype upon loss of Epstein-Barr virus (EBV) genomes in vitro is evidence for a viral contribution to tumor maintenance despite the tightly restricted pattern of EBV gene expression in BL. Akata cells retaining virus manifest increased resistance to apoptosis under growth limiting conditions, although ambiguity exists regarding the exact mechanisms involved. By examining global cellular gene expression differences in Akata subclones that had either retained or lost EBV, we identified spermidine/spermine N1-acetyltransferase (SSAT), an inducible acetylating enzyme whose catabolism of polyamines affects both apoptosis and cell growth, as one of a limited number of cellular genes up-regulated upon loss of EBV. Keywords: Disease state analysis
Project description:Epstein-Barr virus is associated with several human malignancies, including Burkitt Lymnphoma. The virus encodes more than 40 microRNAs, which participate in its possible pathogenetic role. We used microarrays to study the effect of the expression of an Epstein-Barr virus-encoded microRNA (ebv-BART6-3p) on the global gene expression profile of Burkitt Lymphoma cell lines.
Project description:Transition of Akata Burkitt lymphoma (BL) cells from a malignant to nonmalignant phenotype upon loss of Epstein-Barr virus (EBV) genomes in vitro is evidence for a viral contribution to tumor maintenance despite the tightly restricted pattern of EBV gene expression in BL. Akata cells retaining virus manifest increased resistance to apoptosis under growth limiting conditions, although ambiguity exists regarding the exact mechanisms involved. By examining global cellular gene expression differences in Akata subclones that had either retained or lost EBV, we identified spermidine/spermine N1-acetyltransferase (SSAT), an inducible acetylating enzyme whose catabolism of polyamines affects both apoptosis and cell growth, as one of a limited number of cellular genes up-regulated upon loss of EBV. Keywords: Disease state analysis We used Affymetrix microarrays to examine variations in global gene expression between the paired EBV-positive (1B6) and EBV-negative (2A8) Akata Burkitt's lymphoma clones. EBV-negative Akata clones were generated by treatment with hydroxyurea, an inhibitor of ribonucleotide reductase that forced rapid loss of EBV episomes and compared to their EBV-positive counterparts. Logarithmically growing EBV-negative and positive Akata EBV clones were seeded at 2.5x10^5 cells/ml in RPMI1640 supplemented with 10% fetal bovine serum, 2mM glutamine, and 100units/ml penicillin/streptomycin. The next day, the cells were incubated for 4 hours in media containing 1% fetal bovine serum (low serum), 2mM glutatmine and 100units/ml penicillin/streptomycin to begin selection of the Akata tumor phenotype. The short incubation time was intended to minimize cell death and other potential downstream effects of low serum treatment, while at the same time initiating expression changes contributing to the tumorigenic phenotype.
Project description:Epstein-Barr virus positive Burkitt's lymphoma cell line Akata (+) and it's EBV-depleted subclone Akata (-) were analyzed for human circRNA expression.
Project description:Epstein-Barr virus (EBV) reactivation in latently infected B cells is essential for persistent infection and B cell receptor (BCR) activation is a physiologically relevant stimulus for EBV reactivation. Post-translational modifications, such as phosphorylation and ubiquitination, are known to be regulated by antigen binding to BCR within minutes. However, a detailed understanding of the signaling alterations at later time when EBV is being actively replicated remains elusive. To gain insights into BCR activation-mediated reprogramming of the cellular environment in both Akata-BX1 (EBV+) and Akata-4E3 (EBV-) B cells, we utilized a 3-plex stable isotope labeling by amino acid in cell culture (SILAC)-based quantitative proteomic approach to monitor the dynamic changes of protein ubiquitination during the course of immunoglobulin G (IgG) cross-linking of BCRs. We observed temporal alterations in the level of ubiquitination on approximately 150 sites in both Akata-BX1 (EBV+) and Akata-4E3 (EBV-) B cells post-IgG cross-linking compared with no cross-linking controls, with the majority of protein ubiquitination down-regulated. Our analysis revealed that IgG cross-linking plays a major role in the regulation of protein ubiquitination in both EBV+ and EBV- B cells. Bioinformatic analyses of up-regulated ubiquitination events revealed significant enrichment of proteins involved in RNA processing. Among the down-regulated ubiquitination events are proteins enriched in apoptosis and the ubiquitin-proteasome pathway. The comparative and quantitative studies provide a foundation for further understanding how BCR activation regulates cellular protein ubiquitination and how EBV utilizes or subverts BCR engagement-mediated changes to facilitate viral replication.
Project description:To gain insights into how EBV latency is maintained, we performed a human genome-wide CRISPR screen in latently EBV-infected Burkitt lymphoma B-cells. Our analyses identified a network of host factors that repress EBV lytic reactivation, centered on the transcription factor MYC and including cohesins, FACT, STAGA and Mediator. RNAseq was used to identify host and viral transcriptome changes in P3HR-1 Burkitt lymphoma cells expressing control, smc1a, supt16h, med12, or tada2b sgRNAs. RNAseq was used to identify host and viral transcriptome changes in Akata EBV+ burkitt lymphoma cells expressing control or myc sgRNAs.
Project description:We used Precision Nuclear Run-on followed by Deep Sequencing (PRO-Seq) to investigate RNA Polymerase (Pol) activity during Epstein-Barr Virus (EBV) reactivation in EBV positive Burkitt's lymphoma cell lines Mutu-I and Akata. Nuclei were harvested from latent cells and after treatment with NaB/TPA (Mutu-I) or anti-IgG (akata) to stimulate reactivation at 1 and 4 and 12h. We identified multiple sites on the EBV genome enriched with Pol displaying distinct patterns of activity, which showed an association with CTCF and open chromatin.
Project description:RNA-seq was used to characterize the LMP1 TES domain-mediated regulation of B-cell genome wide target genes. We created an inducible stable EBV-negative Akata Burkitt Lymphoma cell line expressing LMP1 wildtype, TES1 functional only, TES2 functional only and TES1/2 non-functional.
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).
Project description:RNAseq was used to identify host and EBV viral transcriptome changes in CHAF1B knock-out Akata EBV+ cells. CHAF1B KO Akata EBV+ cells were subjected to RNAseq analysis. The Akata EBV+ cells expressing control sgRNA was used as the control.