EBV-encoded microRNAs Research Articles

EBV-Encoded MicroRNA-BART17-3p Targets DDX3X and Promotes EBV Infection in EBV-Associated T/Natural Killer-Cell Lymphoproliferative Diseases.

Epstein-Barr virus (EBV) persistently infects T/natural killer (NK) cells causing an array of refractory EBV-associated T/NK-cell lymphoproliferative disorders. EBV-encoded microRNAs are important regulators for EBV latent infection and tumorigenesis. However, the roles of most EBV microRNAs in EBV-infected T/NK cells remain poorly understood. On the basis of a search of the doRiNA database and the BiBiServ2-RNAhybrid website, we predicted that EBV-miR-BART17-3p targeted DDX3X, and we verified the hypothesis by dual-luciferase reporter assay and cell function experiments. In addition, we collected 50 EBV-positive T-, B-, and NK-cell samples from the peripheral blood of EBV-positive cases to examine the role of EBV-miR-BART17-3p in the disease. We found that EBV-miR-BART17-3p directly targeted DDX3X and downregulated DDX3X expression. By analyzing EBV-positive cell samples from cell lines and patients, we found that EBV-miR-BART17-3p was highly expressed only in EBV-positive NK cells and that the overexpression was significantly related to high EBV loads in EBV-infected NK cells. Furthermore, we found that EBV-miR-BART17-3p downregulated the RIG-I-like receptor antiviral pathway and promoted the expression of EBV-encoded proteins in EBV-infected NK cells by targeting DDX3X. Our study showed that EBV-miR-BART17-3p was abundantly expressed in EBV-infected NK cells and inhibited the important antivirus immune responses of hosts by targeting DDX3X of the RIG-I-like receptor pathway. These findings could help us gain insights into the pathogenic mechanisms underlying EBV-associated T/NK-cell lymphoproliferative disorders and find the potential therapeutic target.

The role of EBV-encoded miRNA in EBV-associated gastric cancer.

Epstein-Barr virus (human herpesvirus 4, EBV) is a linear double-stranded DNA virus that infects over 90% of the population worldwide. However, our understanding of EBV's contribution to tumorigenesis of EBV-associated GC (EBVaGC) remains incomplete. Recent advancements in EBVaGC research have highlighted that EBV-encoded microRNAs (miRNAs) play prominent roles in critical cellular processes such as migration, cell cycle, apoptosis, cell proliferation, immune response, and autophagy. Notably, the largest group of EBV-encoded miRNAs, known as BamHI-A rightward transcripts (BARTs), exhibit bidirectional effects in EBVaGC. For instance, they present both anti-apoptotic and pro-apoptotic functions and enhance chemosensitivity while also conferring resistance to 5-fluorouracil. Despite these findings, the comprehensive mechanisms through which miRNAs contribute to EBVaGC are yet to be fully elucidated. In this work, we summarize the current evidence of the roles of miRNA in EBVaGC, particularly with the application of multi-omic techniques. Additionally, we discuss the application of miRNA in EBVaGC in retrospective analyses and provide novel perspectives on the use of miRNA in EBVaGC in translational medicine.

Diverse Expression Patterns of EBV Oncogenes (LMP2A, EBV-Encoded microRNA, and EBV-encoded dUTPase) in EBV Associated Gastric Carcinoma and their Association with Viral Loads.

The chromogenic in situ hybridization (CISH) test is the gold standard for detecting Epstein-Barr virus (EBV)-associated gastric carcinoma (GC). Real-time (RT) PCR method is also a sensitive test that can detect the viral load in samples. As such, three EBV oncogenes were investigated in this study. RNA extraction and cDNA synthesis were performed on GC tissues of nine patients, who were previously confirmed to have EBVGC subtype. In addition, 44 patients that had positive RT-PCR but negative CISH results were also included as the control group. TaqMan RT-PCR analysis was performed to determine the expression of EBV-encoded microRNAs, and the expression of EBV-encoded dUTPase, as well as LMP2A, was analyzed by SYBR Green RT-PCR. EBV-encoded microRNAs and LMP2A were identified in 2 out of 9 (22%) EBVGC subtypes. In addition, EBV-encoded dUTPase was detected in 4 out of 9 (44.5%) EBVGC subtypes. EBV-encoded dUTPase was also expressed in a sample of the control group. The expression of LMP2A, EBV-encoded microRNAs, and EBV-encoded dUTPase viral oncogenes in patients with high EBV viral loads indicates that these expressions correlate with viral loads. Our findings indicate that the EBV-encoded dUTPase gene may have a role in EBVGC patients' non-response to treatment and might be considered a Biomarker-targeted therapy.

Regulation of B cell receptor signalling by Epstein-Barr virus nuclear antigens.

The cancer-associated Epstein-Barr virus (EBV) latently infects and immortalises B lymphocytes. EBV latent membrane protein 2A and EBV-encoded microRNAs are known to manipulate B cell receptor signalling to control cell growth and survival and suppress lytic replication. Here, we show that the EBV transcription factors EBNA2, 3A, 3B and 3C bind to genomic sites around multiple B cell receptor (BCR) pathway genes, regulate their expression and affect BCR signalling. EBNA2 regulates the majority of BCR pathway genes associated with binding sites, where EBNA3 proteins regulate only 42% of targets predicted by binding. Both EBNA2 and 3 proteins predominantly repress BCR pathway gene expression and target some common genes. EBNA2 and at least one EBNA3 protein repress the central BCR components CD79A and CD79B and the downstream genes BLNK, CD22, CD72, NFATC1, PIK3CG and RASGRP3. Studying repression of CD79B, we show that EBNA2 decreases transcription by disrupting binding of Early B cell Factor-1 to the CD79B promoter. Consistent with repression of BCR signalling, we demonstrate that EBNA2 and EBNA3 proteins suppress the basal or active BCR signalling that culminates in NFAT activation. Additionally, we show that EBNA2, EBNA3A and EBNA3C expression can result in reductions in the active serine 473 phosphorylated form of Akt in certain cell contexts, consistent with transcriptional repression of the PI3K-Akt BCR signalling arm. Overall, we identify EBNA2, EBNA3A and EBNA3C-mediated transcription control of BCR signalling as an additional strategy through which EBV may control the growth and survival of infected B cells and maintain viral latency.

The Epstein-Barr Virus Enhancer Interaction Landscapes in Virus-Associated Cancer Cell Lines.

Epstein-Barr virus (EBV) persists in human cells as episomes. EBV episomes are chromatinized and their 3D conformation varies greatly in cells expressing different latency genes. We used HiChIP, an assay which combines genome-wide chromatin conformation capture followed by deep sequencing (Hi-C) and chromatin immunoprecipitation (ChIP), to interrogate the EBV episome 3D conformation in different cancer cell lines. In an EBV-transformed lymphoblastoid cell line (LCL) GM12878 expressing type III EBV latency genes, abundant genomic interactions were identified by H3K27ac HiChIP. A strong enhancer was located near the BILF2 gene and looped to multiple genes around BALFs loci. Perturbation of the BILF2 enhancer by CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) altered the expression of BILF2 enhancer-linked genes, including BARF0 and BALF2, suggesting that this enhancer regulates the expression of linked genes. H3K27ac ChIP followed by deep sequencing (ChIP-seq) identified several strong EBV enhancers in T/NK (natural killer) lymphoma cells that express type II EBV latency genes. Extensive intragenomic interactions were also found which linked enhancers to target genes. A strong enhancer at BILF2 also looped to the BALF loci. CRISPRi also validated the functional connection between BILF2 enhancer and BARF1 gene. In contrast, H3K27ac HiChIP found significantly fewer intragenomic interactions in type I EBV latency gene-expressing primary effusion lymphoma (PEL) cell lines. These data provided new insight into the regulation of EBV latency gene expression in different EBV-associated tumors. IMPORTANCE EBV is the first human DNA tumor virus identified, discovered over 50 years ago. EBV causes ~200,000 cases of various cancers each year. EBV-encoded oncogenes, noncoding RNAs, and microRNAs (miRNAs) can promote cell growth and survival and suppress senescence. Regulation of EBV gene expression is very complex. The viral C promoter regulates the expression of all EBV nuclear antigens (EBNAs), some of which are very far away from the C promoter. Another way by which the virus activates remote gene expression is through DNA looping. In this study, we describe the viral genome looping patterns in various EBV-associated cancer cell lines and identify important EBV enhancers in these cells. This study also identified novel opportunities to perturb and eventually control EBV gene expression in these cancer cells.

Virus-Mediated Inhibition of Apoptosis in the Context of EBV-Associated Diseases: Molecular Mechanisms and Therapeutic Perspectives.

Epstein-Barr virus (EBV), the representative of the Herpesviridae family, is a pathogen extensively distributed in the human population. One of its most characteristic features is the capability to establish latent infection in the host. The infected cells serve as a sanctuary for the dormant virus, and therefore their desensitization to apoptotic stimuli is part of the viral strategy for long-term survival. For this reason, EBV encodes a set of anti-apoptotic products. They may increase the viability of infected cells and enhance their resistance to chemotherapy, thereby contributing to the development of EBV-associated diseases, including Burkitt’s lymphoma (BL), Hodgkin’s lymphoma (HL), gastric cancer (GC), nasopharyngeal carcinoma (NPC) and several other malignancies. In this paper, we have described the molecular mechanism of anti-apoptotic actions of a set of EBV proteins. Moreover, we have reviewed the pro-survival role of non-coding viral transcripts: EBV-encoded small RNAs (EBERs) and microRNAs (miRNAs), in EBV-carrying malignant cells. The influence of EBV on the expression, activity and/or intracellular distribution of B-cell lymphoma 2 (Bcl-2) protein family members, has been presented. Finally, we have also discussed therapeutic perspectives of targeting viral anti-apoptotic products or their molecular partners.

Identification of multiple sclerosis-related genes regulated by EBV-encoded microRNAs in B cells

BackgroundMultiple sclerosis (MS) is driven by the interaction between genetic susceptibility and environmental triggers, particularly to Epstein-Barr virus (EBV) infection. EBV-encoded microRNAs (miRNAs) are abundantly expressed in all stages of EBV infection and latency, which can target both viral and host cellular mRNAs, allowing EBV-infected B cells to evade the host immune response. However, it remains a big gap to understand the roles of EBV miRNAs and their target genes in MS pathogenesis. MethodsWe investigated the correlation between MS-related viruses infection and MS risk quantitatively by systematic analysis. All MS-related genes in B cells were obtained by integrating MS susceptibility genes and differentially expressed genes from B cells. In comparison with differentially expressed genes from B cells after EBV infection in vitro, we confirmed EBV-regulated, MS-related genes. Subsequently, we obtained target EBV miRNAs which can regulate these genes from several online databases. By constructing pathway-pathway, pathway-gene and protein-protein interaction networks, we further screened out MS-related genes and risk pathways regulated by EBV miRNAs. Finally, we identified target EBV miRNAs which may directly regulate MS-related genes through bioinformatic prediction. ResultsEBV infection showed the strongest correlation with MS risk. A total of 568 MS-related genes and 80 risk pathways in B cells were obtained. We then identified 112 MS-related genes and 18 associated risk pathways that EBV was involved in. In addition, 33 human target genes regulated by 33 EBV miRNAs overlapped with EBV-regulated, MS-related genes. Finally, 15 target EBV miRNAs and their regulated, 7 MS-related genes (MALT1, BCL10, IFNGR2, STAT3, CXCR4, PTK2B and FOXP1) have been confirmed as crucial pathogenic molecules, which could promote the initiation and development of MS through NF-kappa B (MALT1 and BCL10) and PD-L1/PD-1 (IFNGR2 and STAT3) pathways. Surprisingly, ebv-miR-BHRF1-2-5p directly targeting MALT1 was confirmed by our experiments, and FOXP1 was identified as a target gene of ebv-miR-BART11. ConclusionsThis work identified the target EBV miRNAs and their regulated, MS-related genes as well as risk pathways, which may provide a novel insight into discovering diagnostic biomarkers and therapeutic targets for MS.

MicroRNA and Other Non-Coding RNAs in Epstein-Barr Virus-Associated Cancers.

Simple SummaryEpstein–Barr virus (EBV) is associated with a variety of malignancies. In this review, we discuss EBV-encoded microRNAs and ncRNAs and consider how their detection could aid in the diagnosis, prognostication, and monitoring of treatment in patients with EBV-associated malignancies, including classical Hodgkin’s lymphoma (cHL), Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL), nasopharyngeal carcinoma (NPC), and gastric carcinoma (GC).EBV is a direct causative agent in around 1.5% of all cancers. The oncogenic properties of EBV are related to its ability to activate processes needed for cellular proliferation, survival, migration, and immune evasion. The EBV latency program is required for the immortalization of infected B cells and involves the expression of non-coding RNAs (ncRNAs), including viral microRNAs. These ncRNAs have different functions that contribute to virus persistence in the asymptomatic host and to the development of EBV-associated cancers. In this review, we discuss the function and potential clinical utility of EBV microRNAs and other ncRNAs in EBV-associated malignancies. This review is not intended to be comprehensive, but rather to provide examples of the importance of ncRNAs.

Landscape of EBV-positive gastric cancer.

Epstein-Barr virus-positive gastric cancer [EBV (+) GC] is associated with EBV infection and is one of the GC subtypes defined by the Cancer Genome Atlas. EBV (+) GC has several distinct genomic or epigenomic features and clinicopathological characteristics compared with other molecular subtypes of GC. Here, we summarize the unique features of EBV (+) GC including the clinical and histopathological features, and discuss associated genetic and epigenetic aberrations. We also discuss noncoding RNAs [EBV-encoded RNAs and EBV-encoded microRNAs (miRNAs)] derived from EBV-infected cells, which have not been described in detail previously. These noncoding RNAs are defined by their roles; for example, EBV-encoded miRNAs play pivotal roles in oncogenesis and tumor progression in EBV (+) GC. We also discuss recent advances in therapeutic modalities for EBV (+) GC, as well as the potential of EBV infection as a predictive biomarker of the response to anti-PD-1 therapy with immune checkpoint inhibitors. We introduce our recent studies focusing on AT-rich interactive domain 1A gene mutations and programmed death ligand-1 overexpression/CD274 copy-number amplification, which are recurrently identified in EBV (+) GC. Finally, based on those findings, we propose potential therapeutic options using candidate-targeted therapies against EBV (+) GC.

Abstract LB211: Epstein-Barr virus BART lncRNAs induce IKZF3/Aiolos to maintain EBV latency and promote tumorigenicity in nasopharyngeal carcinoma

Abstract Epstein-Barr virus (EBV) infection is one of the main causative factors of nasopharyngeal carcinoma (NPC), which is prevalent in southern China and some Southeast Asian countries. EBV infection in NPC cells is characteristically type II latency, where very few viral proteins (EBNA1, LMP1 and LMP2A) but high levels of noncoding RNAs (especially BARTs: BamHI-A rightward transcripts) are expressed. Our previous study found that BART lncRNAs modulate transcription of IKZF3, which encodes Aiolos, through epigenetic pathways in NPC cells. However, the biological significance functional mechanism of IKZF3/Aiolos are not yet clearly defined. We now further show that IKZF3/Aiolos expression levels are associated with copy numbers of EBV episomes in the EBV-harboring NPC cell line, NPC43, and specifically correlate to the level of lnc-BARTs, as demonstrated using CRISPR systems. Transcriptomic analysis of IKZF3-knockout C666-1 NPC cells revealed that 17 EBV-encoded microRNAs and 53 lytic genes were significantly downregulated or upregulated (FDR p value <0.05 and posterior fold changes >1.5). Immunoblot testing showed that the EBV lytic reactivator Zta and antigen BMRF1 are upregulated following knockout of IKZF3 in C666-1 cells, suggesting involvement of IKZF3/Aiolos in maintaining EBV latency in NPC. Gene ontology and KEGG (Kyoto Encyclopaedia of Genes and Genomes) analysis of IKZF3-knockout C666-1 cells found that most of the host genes with differential expression that are enriched in Epstein-Barr virus infection are associated with apoptosis, focal adhesion, cancer cell growth and mammary neoplasm (p value <0.01). In addition, a soft agar assay demonstrated that oncogenic transformation of C666-1 cells was attenuated after knocking down expression of lnc-BARTs, knocking out IKZF3, or treatment with the IKZF3 inhibitor lenalidomide. Immunoprecipitation (IP) testing showed that Aiolos interacted with core elements (HDAC1 and MTA2) of the Mi-2/NuRD (nucleosome remodelling deacetylase) complex in NPC cells. Our findings suggest that Aiolos may recruit Mi-2/NuRD complexes for deacetylation of histone lysine residues bordering promoter regions of EBV lytic genes and host tumour suppressor genes, leading to inhibition of reactivation of EBV lytic infection, thereby preventing apoptosis of NPC cells. Citation Format: Songtao HE, Jiayan Liu, Honglin Chen. Epstein-Barr virus BART lncRNAs induce IKZF3/Aiolos to maintain EBV latency and promote tumorigenicity in nasopharyngeal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB211.

Epstein-Barr virus-encoded microRNAs involve in tumorigenesis and development.

Epstein-Barr virus (EBV), a definite tumorigenic virus, is closely related to the development of nasopharyngeal cancer, gastric cancer, lymphoma and other tumors. EBV encodes a total of 44 mature microRNAs, which can regulate the expression of virus and host genes. EBV-encoded microRNAs and their regulated target molecules participate in the biological functions of tumor apoptosis, proliferation, invasion, and metastasis during tumorigenesis and development, and play an important role in the development of tumor.

A review on EBV encoded and EBV-induced host microRNAs expression profile in different lymphoma types.

Previous literature supports the variations in microRNAs expression levels among lymphoma patients due to EBV infection. These alterations can be observed in both EBV-encoded-microRNAs and EBV-induced cellular microRNAs. Moreover, changes in the microRNA profile could be significant in disease progression. This study aimed to assess published literature to obtain a microRNA profile for both EBV-encoded microRNAs and EBV-induced cellular microRNAs among lymphoma patients. We searched common available electronic databases by using relevant keywords. The result demonstrated that EBV infection could alter the microRNA expression levels among lymphoma patients. In Burkitt lymphoma, hsa-miR197 and miR510 were most frequently assessed human micro RNAs. Also, miR-BART6-3P and miR-BART17-5P were the most frequent viral micro RNAs in Burkitt lymphoma. Other human important micro RNAs were hsa-miR155 (in Diffuse large B cell lymphoma (DLBCL)), hsa-miR145 (in Nasal natural killer T cell lymphoma (NNKTCL)), miR-96, miR-128a, miR-128b, miR-129, and miR-205 (in Classic Hodgkin lymphoma (CHL)), miR-21, miR-142-3P, miR-126, miR-451 and miR-494-3P (in Nasal natural killer cell lymphoma (NNKCL)). Also, viral assessed micro RNAs were miR-BART1-5P (in DLBCL and NNKTCL), miR-BART-5 (in CHL), and EBV-miR-BART20-5P (in NNKCL). In conclusion, it could be suggested that EBV-encoded-microRNAs and EBV-induced cellular-microRNAs can be utilized as helpful factors for different types of lymphoma diagnoses or prognostic factors. Moreover, the mentioned microRNAs can also be promising therapeutic targets and can be used to modulate the oncogenes.

EBV-miR-BART10-3p and EBV-miR-BART22 promote metastasis of EBV-associated gastric carcinoma by activating the canonical Wnt signaling pathway.

Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC) constitutes the largest subpopulation in EBV-associated tumors worldwide. To date, 44 mature EBV-encoded microRNAs (EBV miRNAs) have been identified, but their roles in EBVaGC development are still poorly understood. In this study, we aimed to investigate the roles and targets of ebv-miR-BART10-3p (BART10-3p) and ebv-miR-BART22 (BART22) in EBVaGC. EBV miRNA expression in EBVaGCs was evaluated by deep sequencing and qRT-PCR, and relationships between BART10-3p or BART22 expression and clinicolpathological characteristics and survival rates of patients with EBVaGC were analyzed. The roles of BART10-3p and BART22 and their underlying mechanisms were further investigated through exogenous overexpression or silencing in EBVaGC cells, and validated in clinical EBVaGC tissue samples. BART10-3p and BART22 were found to be highly expressed in the EBVaGC cell lines SNU719 and YCCEL1. Higher expression of BART10-3p or BART22 in primary EBVaGC samples was significantly associated with lymph node metastasis and a worse 5-year overall survival. BART10-3p and BART22 promoted cell migration and invasion by targeting adenomatous polyposis coli (APC) and Dickkopf 1 (DKK1), thereby activating the Wnt signaling pathway and, consequently, upregulating downstream Twist and downregulating downstream E-cadherin. In 874 primary gastric carcinoma samples, APC and DKK1 were found to be lower expressed in EBVaGC than in EBV-negative samples, and their expression levels were inversely correlated with those of BART10-3p and BART22 in 71 EBVaGC samples. From our data we conclude that BART10-3p and BART22 play vital roles in promoting EBVaGC metastasis by targeting APC and DKK1 and, subsequently, activating the Wnt signaling pathway, thereby providing novel prognostic biomarkers and potential therapeutic targets for EBVaGC.

Circulating Epstein-Barr virus microRNA profile reveals novel biomarker for nasopharyngeal carcinoma diagnosis.

Nasopharyngeal carcinoma (NPC), a tumor quite prevalent in Asia, is closely associated with Epstein-Barr virus (EBV) infection status. Many NPC patients are not able to be treated in time when being diagnosed at an advanced stage. EBV-encoded microRNAs are reliable sources of biomarkers for NPC diagnosis. In this study, we conducted circulating EBV microRNAs profiling by quantitative reverse transcription polymerase chain reaction (qRT-PCR) among plasma samples of 159 NPC patients versus 145 normal controls (NCs) and serum samples of 60 NPC patients versus 60 NCs. Among the 44 mature EBV-encoded miRNAs, only miR-BART19-3p in plasma was proved to be significantly up-regulated in NPC patients (P< 0.05; fold change (FC) > 2.0). The area under the receiver operating characteristic curve (AUC) for the signature to discriminate NPC patients from NCs was 0.848 with the sensitivity and specificity being 71.7% and 72.3%, respectively. The identified biomarker was analyzed in tissue specimens (44 NPC VS. 32NCs) and proved to be consistently up-regulated in NPC tumor tissues. Bioinformatics analysis was further conducted to predict the potential targets of miR-BART-19-3p, which provided some hints to its close relationship with NPC development. In conclusion, we identified a novel biomarker- plasma miR-BART19-3p for the detection of NPC.

High Levels of Class I Major Histocompatibility Complex mRNA Are Present in Epstein-Barr Virus-Associated Gastric Adenocarcinomas.

Epstein–Barr virus (EBV) is responsible for approximately 9% of stomach adenocarcinomas. EBV-encoded microRNAs have been reported as reducing the function of the class I major histocompatibility complex (MHC-I) antigen presentation apparatus, which could allow infected cells to evade adaptive immune responses. Using data from nearly 400 human gastric carcinomas (GCs), we assessed the impact of EBV on MHC-I heavy and light chain mRNA levels, as well as multiple other components essential for antigen processing and presentation. Unexpectedly, mRNA levels of these genes were as high, or higher, in EBV-associated gastric carcinomas (EBVaGCs) compared to normal control tissues or other GC subtypes. This coordinated upregulation could have been a consequence of the higher intratumoral levels of interferon γ in EBVaGCs, which correlated with signatures of increased infiltration by T and natural killer (NK) cells. These results indicate that EBV-encoded products do not effectively reduce mRNA levels of the MHC-I antigen presentation apparatus in human GCs.

EBV encoded miRNA BART8-3p promotes radioresistance in nasopharyngeal carcinoma by regulating ATM/ATR signaling pathway.

Resistance to radiotherapy is one of the main causes of treatment failure in patients with nasopharyngeal carcinoma (NPC). Epstein-Barr virus (EBV) infection is an important factor in the pathogenesis of NPC, and EBV-encoded microRNAs (miRNAs) promote NPC progression. However, the role of EBV-encoded miRNAs in the radiosensitivity of NPC remains unclear. Here, we investigated the effects of EBV-miR-BART8-3p on radiotherapy resistance in NPC cells in vitro and in vivo, and explored the underlying molecular mechanisms. Inhibitors of ataxia telangiectasia mutated (ATM)/ataxia telangiectasia mutated and Rad3-related (ATR) (KU60019 and AZD6738, respectively) were used to examine radiotherapy resistance. We proved that EBV-miR-BART8-3p promoted NPC cell proliferation in response to irradiation in vitro and associated with the induction of cell cycle arrest at the G2/M phase, which was a positive factor for the DNA repair after radiation treatment. Besides, EBV-miR-BART8-3p could increase the size of xenograft tumors significantly in nude mice. Treatment with KU60019 or AZD6738 increased the radiosensitivity of NPC by suppressing the expression of p-ATM and p-ATR. The present results indicate that EBV-miR-BART8-3p promotes radioresistance in NPC by modulating the activity of ATM/ATR signaling pathway.

The roles of EBV-encoded microRNAs in EBV-associated tumors.

Epstein-Barr virus (EBV) is believed to be a pathogen causing a number of human cancers, but the pathogenic mechanisms remain unclear. An increasing number of studies have indicated that EBV-encoded microRNAs (EBV miRNAs) are expressed in a latency type- and tumor type-dependent manner, playing important roles in the development and progression of EBV-associated tumors. By targeting one or more genes of the virus and the host, EBV miRNAs are responsible for the deregulation of a variety of viral and host cell biological processes, including viral replication, latency maintenance, immune evasion, cell apoptosis and metabolism, and tumor proliferation and metastasis. In addition, some EBV miRNAs can be used as excellent diagnostic, prognostic and treatment efficacy predictive biomarkers for EBV-associated tumors. More importantly, EBV miRNA-targeting therapeutics have emerged and have been developing rapidly, which may open a new era in the treatment of EBV-associated tumors in the near future.

Regulation of necroptotic factors in gastric cancer cells through miR-93-5p and EBV-miR-BART 18-3p.

Abstract Epstein-Barr virus (EBV), as well as Helicobacter pylori, has recently been admitted as an infective agent causing gastric carcinoma(GC). EBV encodes multiple non-coding RNAs during all types of latency, 44 mature miRNAs have been identified. EBV-encoded microRNAs showed that these small molecules function as post-transcriptional gene regulators and may play a role in the carcinogenesis process. MiR-BARTs produced by EBV can regulate cellular genes for preventing apoptosis and escaping the host immune system. We examined interactions of microRNAs with necroptosis factors in EBV positive/negative gastric cancer cells. RIPKs(receptor-interacting serine/threonine-protein kinase) were little expressed in EBV positive cells. Incubation of EBV negative gastric cells with EBV particles or exosome purified from EBV positive cells revealed that MLKL as a regulator of necroptosis were decreased. We investigated expression profiles for viral and cellular miRNAs in EBV positive/negative gastric cancer cells. To identify possible miRNAs targeting necrotic factors, we searched miRNA candidates that could bind to sequence in the 3′UTR of necrosis factors using an online miRNA database. We found the putative target binding sites of EBV-miR-BART18-3p (BART18-3p) within MLKL 3′UTR and miR-93-5p within RIPK3 3′UTR. Expectedly, we observed that EBV negative KATOIII transfected with mimics for BART18-3p diminished expression of p-MLKL, inhibitor for BART18-3p increased p-MLKL in EBV positive SNU719. However, mimics for miR-93-5p decreased RIPK1 rather than RIPK3 in EBV negative KATOIII. Taken together, we suggested that EBV might regulate necroptosis in gastric cancer cells through miRNAs including BART18-3p and miR-93-5p.

Nasopharyngeal brushing: a convenient and feasible sampling method for nucleic acid-based nasopharyngeal carcinoma research

BackgroundTissue specimens for nasopharyngeal carcinoma (NPC) research are scarce because of sampling difficulties. Previous studies have suggested non-invasive nasopharyngeal brushing as an effective sampling method for NPC diagnosis. The present study aimed to evaluate the feasibility of nasopharyngeal brushing in the acquisition of NPC nucleic acids for research.MethodsNasopharyngeal brushing samples were acquired from 24 healthy individuals and 48 NPC patients. Tissues from 48 NPC and 18 nasopharyngitis patients were collected by endoscopic biopsy. The expression levels of tumor suppressor genes (TSGs) and Epstein–Barr virus (EBV)-encoded microRNAs as well as EBV DNA copy number were measured by quantitative polymerase chain reaction in both types of samples.ResultsAmong six TSGs examined, the expression levels of two genes were significantly decreased in nasopharyngeal brushing and tissue samples from NPC patients as compared with those from healthy/nasopharyngitis individuals. Four EBV-encoded microRNAs, mir-bart1-5p, mir-bart5, mir-bart6-5p, and mir-bart17-5p, were significantly up-regulated in both NPC brushing and tissue samples compared with those from healthy/nasopharyngitis controls (P < 0.001). EBV DNA was significantly increased in both nasopharyngeal brushing samples (P < 0.001) and tissue samples (P < 0.001) from NPC patients in comparison with those from healthy controls.ConclusionsNasopharyngeal brushing can obtain sufficient tumoral materials for the analysis of viral nucleic acid, including EBV-encoded microRNAs and EBV DNA. For the detection of TSG expression, nasopharyngeal brushings was feasible but inferior to tissue samples. This study confirms nasopharyngeal brushing as an applicable sampling method that can aid in nucleic acid-based NPC research.

EBV-encoded microRNAs profile evaluation in pediatric liver transplant recipients

BackgroundEpstein–Barr virus (EBV) infects 90% of the world population, commonly causing self-limiting infectious mononucleosis or rarely inciting a range of malignancies. EBV microRNAs (miRNAs) were discovered by sequencing libraries of small RNAs generated from several EBV-positive cell lines. Little is known about their roles, but their high stability and easy quantification make these molecules potential biomarkers. ObjectivesIn this study a stem-loop MGB real-time RT-PCR has been used to detect and quantify miR-BART2-5p, miR-BART15 and miR-BART22 EBV miRNAs levels. Study designsThe profiles of EBV miRNAs levels were evaluated in 51 serum samples of 37 pediatric liver transplant patients subdivided in 3 study groups: EBV seronegative, EBV seropositive and PCR negative, EBV seropositive and PCR positive. ResultsmiR-BART22 serum levels in patients with positive EBV PCR were significantly higher than those in patients with negative EBV PCR (p=0.0005). On the contrary, miR-BART2-5p and miR-BART15 did not exhibit significant difference in positive and negative EBV PCR patients (p=0.5511 and p=0.3523, respectively). ConclusionThis study described a method for quantitative detection of miR-BART 22, miR-BART2-5p and miR-BART15 EBV miRNAs in liver transplanted patients, and suggests the use of miR-BART22 as a potential biomarker for EBV reactivation.