Expression analysis of lung miRNAs responding to ovine VM virus infection by RNA-seq

Background: MicroRNAs (miRNAs) are short endogenous, single-stranded, noncoding small RNA molecules of approximately 22 nucleotides in length. They regulate gene expression posttranscriptionally by silencing mRNA expression, thus orchestrating many physiological processes. The Small Ruminant Lentiviruses (SRLV) group includes the Visna Maedi Virus (VMV) and Caprine Arthritis Encephalitis (CAEV) viruses, which cause a disease in sheep and goats characterized by pneumonia, mastitis, arthritis and encephalitis. Their main target cells are from the monocyte/macrophage lineage. To date, there are no studies on the role of miRNAs in this viral disease. Results: Using RNA-seq technology and bioinformatics analysis, the expression levels of miRNAs during different clinical stages of infection were studied. A total of 212 miRNAs were identified, of which 46 were conserved sequences in other species but found for the first time in sheep, and 12 were completely novel. Differential expression analysis comparing the uninfected and seropositive groups showed changes in several miRNAs; however, no significant differences were detected between seropositive asymptomatic and diseased sheep. The robust increase in the expression level of oar-miR-21 is consistent with its increased expression in other viral diseases. Furthermore, the target prediction of the dysregulated miRNAs revealed that they control genes involved in proliferation-related signalling pathways, such as the PI3K-Akt, AMPK and ErbB pathways. Conclusions: To the best of our knowledge, this is the first study reporting miRNA profiling in sheep in response to SRLV infection. The known functions of oar-miR-21 as a regulator of inflammation and proliferation appear to be a possible cause of the lesions caused in the sheep's lungs. This miRNA could be an indicator for the severity of the lung lesions, or a putative target for therapeutic intervention.This work was supported by a UPV/EHU grant (GIU14/23) provided to B.M. Jugo, two predoctoral fellowships from the UPV/EHU to M. Bilbao-Arribas (PIF17/306) and E. Varela-Martínez (PIF15/361) and a postdoctoral fellowship from the UPV/EHU to Dr. N. Abendaño (ESPDOC16/43)

A Multi-Modal Approach To Sulforaphane-Mediated Chemoprevention Using The Epigenetic Network

Colon cancer is the third most common diagnosed cancer in both women and men in the United States. It is reported that dietary factors are responsible for 70-90% of colon cancer cases. While some investigators have observed a direct correlation in high fat diets and cancer initiation, other studies have observed opposite effects mediated by fruit and vegetable consumption. Recently, studies have highlighted the importance of Epigenetics as it modifies gene expression without changing the DNA sequence. Notably, several nutritive compounds have been reported to alter epigenetic profiles in many cancer forms. SFN, an isothiocyanate found in cruciferous vegetables such as broccoli sprouts and cabbage is reported to modify many carcinogenic genes. Although SFN has been reported to act as a potent HDAC inhibitor, its ability to mediate epigenetic regulation on DNA methylation and non-coding RNAs is severely understudied. Accordingly, in this study we sought to monitor the effects of isothiocyanate SFN on the entire epigenetic network. Using easily attainable and physiologically relevant doses of SFN, we observed a SFN-mediated decrease in cell viability and cell density with a concomitant induction of apoptosis. Additionally, we also observed a significant down-regulation in HDAC1 and hTERT mRNA, protein and activity levels in CRC cells. To further test SFN’s pleotropic abilities on the epigenetic network, we measured DNA methyltransferase (DNMT) and non-coding RNA microRNA-21 (miR-21) levels in CRC cells. For the first time, we showed that physiologically relevant doses of SFN significantly down-regulated the expression of oncogenic miR-21. In addition, we also observed significant down-regulation of DNMT1 and DNMT3B protein levels, as well as regulation of global methylation levels. Interestingly we discovered that a knockdown (KD) of miR-21 resulted in a significant down-regulation of DNMT1 and DNMT3B protein and global methylation levels mediated by a significant increase in phosphatase tensin homolog deleted on chromosome ten (PTEN). Collectively, our studies provide novel findings for safe ways to mediate epigenetic therapy CRC cells

Non-coding RNAs in ovine immunity: Identification of unannotated genes and functional analyses of high throughput genomic data

210 p.Non-coding RNAs (ncRNAs) are involved in several biological processes in mammals, including the immune system response to pathogens and vaccines. The annotation and functional characterization of two of the main classes of ncRNAs, microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), is more advanced in humans than in livestock species, and thus, there is limited knowledge about the function of these transcripts. The main objective of this work was the identification of ovine non-coding genes, concretely miRNA and lncRNA genes, that are involved in the innate and adaptive immune responses induced by vaccines, vaccine components and pathogen infections. For this purpose, high-throughput transcriptome sequencing datasets produced for this purpose and datasets publicly available were analysed with bioinformatic tools and workflows in order to identify unannotated non-coding genes, profile their expression in different tissues and perform evolutionary conservation analyses. More than 12000 unannotated ovine lncRNAs and 1000 ovine miRNAs were identified in the different analyses, with varying levels of sequence conservation. Differential expression analyses between unstimulated samples and samples stimulated with pathogen infection or vaccination resulted in hundreds of lncRNAs and miRNAs with changed expression. Gene co-expression analyses revealed immune gene-enriched clusters associated with immune system activation. These genes make up a prioritized set of potential candidates for deeper experimental analyses. Taken together, these results should help completing the sheep non-coding gene catalogue, and most importantly, they give evidence of immune state-specific ncRNA expression patterns in a livestock species

Investigating mechanisms of Epstein-Barr virus reactivation in epithelial cells

Approximately 10% of all gastric cancer cases worldwide are associated with Epstein-Barr virus (EBV) infection. This subgroup differs from non-infected equivalents through a distinct mutational and epigenetic signature, which is crucial for the establishment and maintenance of the viral latent state. Disruption of the latent state and subsequent induction of the lytic cycle, called reactivation, can be used as a therapeutic strategy for this cancer type through either combination with antiviral agents or by induction of an EBV-specific immune response. In this study, epigenetic inhibitors were tested for their potential to reactivate EBV using a novel screening method based on the expression of Zebra, a key regulator of EBV reactivation. Histone deacetylase inhibitors (HDACis), more specifically benzamide-based HDACis, were the most potent group of reactivating agents in this screen. Those HDACis induced the expression of lytic cycle genes as well as targeted cell killing in combination with antiviral agents. Moreover, the reactivation through those agents was sufficient to activate cytotoxic T-cells and thereby ensured a supportive role of the immune system in targeted cell killing. In an additional screen, topoisomerase inhibitors and other chemotherapeutic agents showed synergistic effects on Zebra expression together with HDACis, which was linked to the induction of p53 expression through those compounds. While p53 is known to be involved in the expression of Zebra, the viral DNA processivity factor Ea-D and the viral DNA binding protein DNBI were identified as a potential novel binding protein of p53 and could thus point towards a novel role of p53 in EBV lytic replication. Taken together, this study identifies potential novel therapeutic compound combinations for the treatment of EBV-associated epithelial cancers that could be translated into the clinic after further assessment

Das nukleäre Antigen 3A von Epstein-Barr Virus und seine Rolle bei der Transformation von B-Lymphozyten

The conversion of Epstein-Barr virus (EBV) infected B lymphocytes to continuously proliferating Lymphoblastoid Cell Lines (LCLs) requires four EBV oncoproteins: EBNA2, EBNALP, EBNA3A, and EBNA3C. EBNA2 and -LP lead to elevated MYC levels, enabling cell proliferation. EBV-driven lymphoblast growth moreover requires the suppression of CDKN2A-mediated cell senescence responses by EBNA3A and -3C. This study describes the genomic landscape of EBNA3A in EBV-infected B cells. ChIP-seq analyses revealed over 10,000 genome-wide EBNA3A binding sites. 65% were located at enhancers and 11% localized to promoters. Target sites included MYC, CDKN2A/B, CCND2, CXCL9/10 and BCL2. EBNA3A sites were co-occupied by BATF-IRF4, ETS-IRF4, RUNX3 as well as other B cell TFs, and clustered into seven unique groups that differed in B cell TF occupancy and histone modifications. EBNA3A ChIP-seq signals increased upon co-occurrence with BATF-IRF4 or RUNX3. The results of ChIP-re-ChIP and shRNA knock-down experiments strongly support a model in which EBNA3A is tethered to DNA through a BATF- and IRF4-containing complex to enable continuous cell proliferation. ChIP-qPCR analyses to investigate the repression of CDKN2A by EBNA3A revealed that EBNA3A directly targets the transcription machinery, reducing RNA Pol II phosphorylation, followed by reduced transcription. EBNA3A moreover mediates the recruitment of the polycomb repressive complex (PRC) 2 in order to facilitate the deposition of repressive H3K27me3 marks across the CDKN2A locus. Chromosome Conformation Capture (3C) assays revealed the existence of a chromatin loop from EBNA3A binding sites to the p14ARF/p16INK4A promoter region, leading to silencing of the CDKN2A locus in order to ensure LCL outgrowth and survival. To further investigate the effects of EBV infection on gene expression, clusters of gene-regulatory sites bound by multiple TFs, so called super-enhancers, were examined in EBV transformed LCLs. This identified ~1800 enhancer sites, co-occupied by four EBV oncoproteins and five NF-κB subunits. 187 of these showed remarkably high and broad H3K27ac signals, characteristic for super-enhancers. They were highly enriched for B cell TF motifs, and often co-occupied by STAT5 and NFAT. EBV super-enhancer-associated genes showed higher expression levels than other LCL genes, and included the oncogenes MYC and BCL2, driving LCL growth and survival. MYC or BCL2 expression could be decreased and LCL growth arrested by disrupting EBV super-enhancers using the bromodomain inhibitor JQ1, or by conditionally inactivating an EBV oncoprotein or NF-κB. These data reveal mechanisms of EBV-induced lymphoproliferation and identify possible therapeutic interventions.Die Umwandlung Epstein-Barr Virus (EBV) infizierter B-Lymphozyten in kontinuierlich proliferierende Lymphoblastoide Zelllinien (LCLs) erfordert vier EBV Onkoproteine: EBNA2, EBNALP, EBNA3A und EBNA3C. EBNA2 und -LP führen zu erhöhten MYC Leveln, was Zellproliferation ermöglicht. EBV-gesteuertes Lymphoblastenwachstum erfordert darüber hinaus die Suppression CDKN2A-vermittelter zellulärer Seneszenzreaktionen durch EBNA3A und -3C. Diese Studie beschreibt das genomische Profil von EBNA3A in EBV-infizierten B-Zellen. ChIP-seq Analysen ergaben genomweit über 10.000 EBNA3A Bindungsstellen. Davon waren 65% in Enhancern und 11% in Promotern lokalisiert. Zielgene umfassten MYC, CDKN2A/B, CCND2, CXCL9/10 sowie BCL2. EBNA3A Bindungsstellen wurden auch von BATF-IRF4, ETS-IRF4, RUNX3 und anderen B-Zell TFs besetzt und unterteilten sich in sieben spezifische Gruppen, die sich in der Besetzung durch B-Zell TFs und Histonmodifikationen unterschieden. EBNA3A ChIP-seq Signale wurden durch gleichzeitige Bindung von BATF-IRF4 oder RUNX3 verstärkt. Die Ergebnisse von ChIP-re-ChIP und shRNA Knockdown Experimenten unterstützen nachhaltig ein Modell in welchem EBNA3A durch einen BATF- und IRF4-enthaltenden Proteinkomplex an DNA gebunden wird, um kontinuierliche Zellproliferation zu gewährleisten. Um die durch EBNA3A vermittelte Repression des CDKN2A Lokus zu untersuchen, wurden ChIP-qPCR Experimente durchgeführt. Diese zeigten, dass EBNA3A direkt am Transkriptionskomplex angreift, um die Phosphorylierung von RNA Pol II zu reduzieren, was zu verminderter Transkription führt. Darüber hinaus vermittelt EBNA3A die Rekrutierung des Polycomb Repressive Complex (PRC) 2, um die Deposition reprimierender H3K27me3 Modifikationen im CDKN2A Gen zu ermöglichen. Chromosome Conformation Capture (3C) Assays bestätigten die Existenz eines Chromatin-Loops von den EBNA3A Bindungsstellen zur p14ARF/p16INK4A Promoterregion, was zur Stilllegung des CDKN2A Gens führt, um folglich das Wachstum und Überleben von LCLs zu gewährleisten. Um den Effekt einer EBV Infektion auf die allgemeine Genexpression zu untersuchen, wurden sogenannte Super-Enhancer, Cluster von Gen-regulatorischen Stellen, an welche eine Vielzahl von TFs gebunden ist, in EBV transformierten LCLs untersucht. Dies ergab etwa 1800 Enhancer,welche gleichzeitig von vier EBV Onkoproteinen und fünf NF-κB Untereinheiten gebunden wurden. 187 davon wiesen außergewöhnlich hohe und breite H3K27ac Signale auf, welche charakteristisch für Super-Enhancer sind. Sie zeigten eine starke Anreicherung von B-Zell TF Motiven und waren oft durch STAT5 oder NFAT gebunden. EBV Super-Enhancer-assoziierte Gene wiesen höhere Expressionslevel als andere LCL Gene auf, und beinhalten die Onkogene MYC und BCL2, welche das Wachstum und Überleben von LCLs vorantreiben. Die Expression von MYC und BCL2 konnte reduziert und das Wachstum von LCLs gestoppt werden, indem die EBV Super-Enhancer entweder durch den Bromodomain-Inhibitor JQ1 inhibiert wurden oder ein EBV Onkoprotein oder NF-κB konditional inaktiviert wurde. Diese Daten legen Mechanismen EBV-induzierter Lymphoproliferation dar und identifizieren neue therapeutische Interventionsmöglichkeiten

Epstein-Barr Virus_Encoded LMP1 Upregulates MicroRNA-21 to Promote the Resistance of Nasopharyngeal Carcinoma Cells to Cisplatin-Induced Apoptosis by Suppressing PDCD4 and Fas-L

<div><p>Approximately 30% of patients with Epstein-Barr virus (EBV)-positive advanced nasopharyngeal carcinoma (NPC) display chemoresistance to cisplatin-based regimens, but the underlying mechanisms are unclear. The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1), a functional homologue of the tumor necrosis factor receptor family, contributes substantially to the oncogenic potential of EBV through the activation of multiple signaling pathways, and it is closely associated with a poorer prognosis for NPC. Recent studies show that EBV infection can induce the expression of many cellular miRNAs, including microRNA-21, a biomarker for chemoresistance. However, neither a link between LMP1 expression and miR-21 upregulation nor their cross talk in affecting chemoresistance to cisplatin have been reported. Here, we observed that stable LMP1-transformed NPC cells were less sensitive to cisplatin treatment based on their proliferation, colony formation, the IC₅₀ value of cisplatin and the apoptosis index. Higher levels of miR-21 were found in EBV-carrying and LMP1-positive cell lines, suggesting that LMP1 may be linked to miR-21 upregulation. These data were confirmed by our results that exogenous LMP1 increased miR-21 in both transiently and stably LMP1-transfected cells, and the knock down of miR-21 substantially reversed the resistance of the NPC cells to cisplatin treatment. Moreover, the proapoptotic factors programmed cell death 4 (PDCD4) and Fas ligand (Fas-L), which were negatively regulated by miR-21, were found to play an important role in the program of LMP1-dependent cisplatin resistance. Finally, we demonstrated that LMP1 induced miR-21 expression primarily by modulating the PI3K/AKT/FOXO3a signaling pathway. Taken together, we revealed for the first time that viral LMP1 triggers the PI3K/Akt/FOXO3a pathway to induce human miR-21 expression, which subsequently decreases the expression of PDCD4 and Fas-L, and results in chemoresistance in NPC cells.</p> </div