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Epstein‐Barr virus (EBV) was the first tumor virus discovered in humans and can cause various types of tumors. Molecular classification suggests that EBV‐associated gastric cancer (EBVaGC) is a unique subtype of gastric cancer.EBV was also the first virus found to encode its own microRNAs. However, the functions of many miRNAs remain unknown. This study investigated the roles and targets of miR‐BART2‐5p (BART2‐5p) and miR‐BART11‐5p (BART11‐5p) in EBVaGC. The expression of RB and p21 in EBVaGC and EBV negative GC (EBVnGC) cells was evaluated by western blotting. Expression of BART2‐5p and BART11‐5p in EBVaGC cells was evaluated by droplet digital PCR. The effects of BART2‐5p or BART11‐5p and their potential mechanisms were further investigated using cell counting kit‐8, colony formation assay, flow cytometry analysis, and transwell assay. BART2‐5p and BART11‐5p were abundantly expressed and RB and p21 were downregulated in EBVaGC cells. BART2‐5p regulates RB and p21 expression by directly targeting them. BART11‐5p regulates RB expression by directly targeting RB. Both BART2‐5p and BART11‐5p promoted proliferation and migration of gastric cancer cells, while inhibiting apoptosis and promoting S‐phase arrest of the cell cycle. Thus, BART2‐5p and BART11‐5p play important roles in promoting proliferation and migration, and inhibiting apoptosis in EBVaGC by targeting RB and p21, thus providing new potential therapeutic targets for EBVaGC. [ABSTRACT FROM AUTHOR]

Aim: The purpose of this study is to investigate the polymorphism and distribution characteristics of BALF2 gene in Epstein–Barr virus (EBV)-associated tumors (gastric cancer, nasopharyngeal carcinoma and lymphoma). Materials & methods: DNA sequences of 349 EBV-related samples were analyzed by nested PCR combined with DNA sequencing. Results: According to the phylogenetic tree, BALF2 was divided into six genotypes (BALF2-A–F). Statistically, the incidence of BALF2-E in nasopharyngeal carcinoma was higher than that in healthy people, and the incidence of BALF2-E in nasopharyngeal carcinoma in South China was higher than that in North China (p = 0.001). Conclusion:BALF2 variants in EBV-associated samples are not only tumor-specific, but also differ between northern and southern regions. [ABSTRACT FROM AUTHOR]

Abstract Background The Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) plays a key role in the B-cell growth transformation by initiating and maintaining the proliferation of infected B-cell upon EBV infection in vitro. Most studies about EBNA-2 have focused on its functions yet little is known for its intertypic polymorphisms. Results Coding region for amino acid (aa) 148-487 of the EBNA-2 gene was sequenced in 25 EBV-associated gastric carcinomas (EBVaGCs), 56 nasopharyngeal carcinomas (NPCs) and 32 throat washings (TWs) from healthy donors in Northern China. Three variations (g48991t, c48998a, t49613a) were detected in all of the samples (113/113, 100%). EBNA-2 could be classified into four distinct subtypes: E2-A, E2-B, E2-C and E2-D based on the deletion status of three aa (294Q, 357K and 358G). Subtypes E2-A and E2-C were detected in 56/113 (49.6%), 38/113 (33.6%) samples, respectively. E2-A was observed more in EBVaGCs samples and subtype E2-D was only detected in the NPC samples. Variation analysis in EBNA-2 functional domains: the TAD residue (I438L) and the NLS residues (E476G, P484H and I486T) were only detected in NPC samples which located in the carboxyl terminus of EBNA-2 gene. Conclusions The subtypes E2-A and E2-C were the dominant genotypes of the EBNA-2 gene in Northern China. The subtype E2-D may be associated with the tumorigenesis of NPC. The NPC isolates were prone harbor to more mutations than the other two groups in the functional domains.

EBV is an important human tumor virus and is closely related to the occurrence of a variety of tumors, involving 10% of gastric cancer. In EBV-associated gastric carcinoma (EBVaGC), EBV expresses restrict viral genes including EBV nuclear antigen 1, EBV encoded small RNAs, Bam HI-A rightward transcripts, latent membrane protein 2A and miRNAs. The role of EBV in gastric carcinogenesis has received increasing attention and is considered to be another pathogenic factor in addition to Helicobacter pylori. A typical characteristic of EBVaGC is the extensive methylation of viral and host genome. Combined with other epigenetic mechanisms, EBV infection acts as an epigenetic driver of EBVaGC oncogenesis. In this review we discuss recent findings of EBV effect on host epigenetic alterations in EBVaGC and its role in oncogenic process. [ABSTRACT FROM AUTHOR]

Epstein‐Barr virus (EBV) is etiologically linked to nasopharyngeal carcinoma, lymphoma, and gastric carcinoma. The aim of this study was to assess the association of TM2 domain containing 3 (TM2D3) and fibroblast growth factor receptor 2 (FGFR2) SNPs rs675436 and rs755793 with susceptibility to EBV‐associated tumors in Chinese Han population. Genomic DNA of 415 patients with cancer and 99 healthy controls was assessed using a MALDI‐TOF mass spectrometer, and was genotyped in EBV‐associated/‐negative gastric cancer (EBVa/nGC), EBV‐associated/‐negative nasopharyngeal carcinoma (EBVa/nNPC), EBV‐associated/‐negative lymphoma (EBVa/nL), and normal controls (NC). The chi‐squared (χ2) test or Fisher's exact test was used to compare all results and P < 0.05 was considered statistically significant. The results showed that the G‐allele of rs675436 was less prevalent in EBVaGC as than in the other groups, while the T‐allele was more prevalent in EBVaGC (EBVaGC vs EBVnGC: P = 0.005, OR = 10.522, 95%CI = 1.377‐80.376; EBVaGC vs NC: P = 0.005, OR = 10.637, 95%CI = 1.392‐81.263). Interestingly, the distribution of the genotype and allele frequencies of rs675436 was significantly different between EBVaGC and EBVaNPC. Neither the genotype nor allele frequency of rs755793 was statistically different between any two groups (P > 0.05). These findings indicated that the TT genotype and T allele frequencies of rs675436 were associated with an increased risk of EBVaGC, while allele A or G of rs755793 had no effect on the occurrence of EBV‐associated tumors in Chinese Han population. [ABSTRACT FROM AUTHOR]

BNLF2a is an Epstein-Barr virus (EBV) immune evasion gene. Its protein is located in the endoplasmic reticulum (ER) membrane, and can inhibit the antigen transporting function of TAP, thereby perturbing the immune response to EBV in lytic and prelatent phase. In order to explore whether the polymorphism of BNLF2a gene has a role in different types of EBV associated tumors, we conducted complete sequencing of the gene BNLF2a in 408 cases of EBV positive tumors (76 lymphomas, 45 gastric carcinomas, and 85 nasopharyngeal carcinomas in northern China and 27 lymphomas, 30 gastric carcinomas, and 57 nasopharyngeal carcinomas in southern China) and throat washings from healthy individuals (39 in northern China and 49 in southern China). Two main variant types of BNLF2a were identified. Type BNLF2a-A, which was similar to B95-8, was dominant in all sub-populations (66.7-100%) in this study. Type BNLF2a-B was characterized by the mutations at position 8 and 40. The variation patterns of BNLF2a were significantly different between samples from northern and southern China ( P < 0.05), and between the tumors and healthy donor samples from the northern China ( P < 0.0167). Type BNLF2a-B was more frequent in healthy donors of northern China (33.3%), and the proportion of this type was higher in the northern than in the southern NPCs. These data demonstrate that the BNLF2a gene is highly conserved, and its polymorphism is geographically restricted. Type BNLF2a-B is more prevalent in northern China and may be less tumor transformative. J. Med. Virol. 87:1946-1952, 2015. © 2015 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Abstract: Epstein-Barr virus (EBV) BZLF1 gene can trigger EBV from latent infection to lytic replicative phase. The functions of BZLF1 are well known, while little is known about its gene polymorphism. In order to elucidate the sequence variations of BZLF1 and its association with malignancies, we analyzed BZLF1 gene in 24 EBV-associated gastric carcinomas, 41 nasopharyngeal carcinomas and 24 throat washing samples from healthy donors in Northern China using PCR-direct sequencing method. Three types and 8 subtypes of BZLF1 were identified. A dominant type BZLF1-A was found in 67 of 89 (75.3%) isolates. Type BZLF1-B was characterized by a common Ala deletion at residue 127, which was detected in 21 of 89 isolates (23.6%). Type BZLF1-C contained only one isolate (GC103), which had the same sequence with the prototype B95-8. Among 3 functional domains of BZLF1 protein, the transactivation domain had most mutations, followed by the bZIP domains (the DNA binding domain and dimerization domain). No prevalence of any subtypes or mutations in the functional domains among three specimen groups was found (P > 0.05). Our study indicates that BZLF1 subtypes and amino acid changes in functional domains are not preferentially associated with EBV-associated gastric carcinomas or nasopharyngeal carcinomas in Northern China. BZLF1 gene variations are geographically restricted rather than tumor-specific polymorphisms. [Copyright &y& Elsevier]

Epstein–Barr virus is a ubiquitous virus and is associated with several human malignances, including the significant subset of gastric carcinoma, Epstein–Barr virus–associated gastric carcinoma. Some Epstein–Barr virus–associated diseases are uniquely prevalent in populations with different geographic origins. However, the features of the disease and geographically associated Epstein–Barr virus genetic variation as well as the roles that the variation plays in carcinogenesis and evolution remain unclear. Therefore, in this study, we sequenced 95 geographically distinct Epstein–Barr virus isolates from Epstein–Barr virus–associated gastric carcinoma biopsies and saliva of healthy donors to detect variants and genes associated with gastric carcinoma and population structure from a genome-wide spectrum. We demonstrated that Epstein–Barr virus revealed the population structure between North China and South China. In addition, we observed population stratification between Epstein–Barr virus strains from gastric carcinoma and healthy controls, indicating that certain Epstein–Barr virus subtypes are associated with different gastric carcinoma risks. We identified that the BRLF1, BBRF3, and BBLF2/BBLF3 genes had significant associations with gastric carcinoma. LMP1 and BNLF2a genes were strongly geographically associated genes in Epstein–Barr virus. Our study provides insights into the genetic basis of oncogenic Epstein–Barr virus for gastric carcinoma, and the genetic variants associated with gastric carcinoma can serve as biomarkers for oncogenic Epstein–Barr virus. [ABSTRACT FROM AUTHOR]

GCNT3 (core 2β-1,6-acetylglucosaminyltransferase) is a novel core mucin synthase. It is known that abnormal expression of GCNT3 promotes the progression of several human cancers. However, its relationship with Epstein-Barr virus (EBV) has not been comprehensively studied. We found GCNT3 expression in EBV-associated gastric cancer cells and tissues to be lower than in EBV-negative gastric cancer cells and tissues, and high expression was significantly associated with advanced tumor-lymph node metastasis. Luciferase reporter assay revealed that miR-BART1-5p directly targeted GCNT3. In addition, miR-BART1-5p mimics transfection was observed to reduce cell proliferation and migration, while miR-BART1-5p inhibitor increased cell proliferation and migration following transfection. In conclusion, both miR-BART1-5p and knockdown of GCNT3 inhibited cell proliferation and migration. In addition, EBV may regulate GCNT3 by affecting the NF-kB signaling pathway. E-cadherin, N-cadherin, vimentin, and p-ERK were found to be downstream molecules of the miR-BART1-5p/GCNT3 pathway. [ABSTRACT FROM AUTHOR]

Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC) is a unique type of gastric carcinomas that promoter hypermethylation of tumor-related genes is extremely frequent to be found. Aquaporin 3 (AQP3) is a small membrane transport protein that plays a crucial role in cancer progression and metastasis. However, there is no experimental study on the expression of AQP3 in EBVaGC and the regulation mechanism of EBV on AQP3. In this study, the loss of AQP3 was contributed by the hypermethylation status of AQP3 promoter in EBVaGC which was caused by elevated expression of DNMT3a. In addition, stable and transient transfection system in SGC7901 showed that viral latent membrane protein 2A (LMP2A) activated phosphorylated ERK and up-regulated DNMT3a. Taken together, LMP2A induced the phosphorylation of ERK, which activated DNMT3a transcription and caused AQP3 expression loss through CpG island methylation of AQP3 promoter in EBVaGC. [ABSTRACT FROM AUTHOR]

EBV-associated gastric carcinoma (EBVaGC) is a specific subgroup of gastric carcinoma, and the multifunctional transcriptional factor NF-κB may contribute to its tumorigenesis. In this study, we comprehensively characterized NF-κB signaling in EBVaGC using qRT-PCR, western blot, immunofluorescence assays, ELISA, and immunohistochemistry staining. NF-κB-signaling inhibitors may inhibit the growth of EBVaGC cells and induce significant apoptosis. IκBα is a key regulatory molecule, and repression of IκBα can contribute to aberrant NF-κB activation. Overexpression of LMP1 and LMP2A in the EBV-negative GC cell line SGC7901 could inhibit the expression of IκBα and induce NF-κB activation. These findings indicate that the canonical NF-κB signal is constitutively activated and plays an important role in EBVaGC tumorigenesis. [ABSTRACT FROM AUTHOR]

As a member of the Bam HI-A rightward transcripts family of the Epstein-Barr virus (EBV), RPMS1 expression has been confirmed in all EBV-associated tumors. However, few studies have investigated the single-nucleotide polymorphisms (SNPs) of RPMS1 , and only one SNP site (g155391a) has been reported to be associated with nasopharyngeal carcinoma occurrence. The objective of this study was to investigate the polymorphism of RPMS1 in EBV-associated tumors (gastric carcinoma, nasopharyngeal carcinoma, and lymphoma). In this research, nested-PCR was performed to analyze DNA sequences of 420 EBV-associated samples. Phylogenetic analysis revealed four RPMS1 genotypes (RPMS1-A, RPMS1-B, RPMS1-C, and RPMS1-D). A significant difference ( p  < 0.05) among northern and southern China samples was observed. Furthermore, there was a significant difference between EBV-associated tumors and healthy controls for RPMS1 ( p  < 0.05). These findings demonstrated that RPMS1 variation was not only tumor-specific but also geographically restricted in EBV-associated samples. [ABSTRACT FROM AUTHOR]

Abstract: The Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1), the only viral protein consistently expressed in all EBV-associated tumors, is classified into five distinct subtypes: P-ala, P-thr, V-leu, V-val and V-pro based on the signature changes at amino acid residue 487. By now, whether the EBNA1 subtypes preferentially associate with particular malignancies or represent geographical polymorphism remains controversial. In China, most studies of the EBNA1 variations focused on nasopharyngeal carcinoma (NPC) in endemic area, among which some suggested the V-val subtype is preferentially associated with NPC. To characterize the variations of EBNA1 in NPC non-endemic area in China and to explore the association of EBNA1 variations with EBV-associated gastric carcinoma (EBVaGC) and NPC, the C-terminal sequences of EBNA1 were analyzed for 41 EBVaGC, 41 NPC biopsies and 55 throat washing (TW) samples from healthy donors in Northern China. Three major patterns of the EBNA1 variations, V-val, P-thrV and V-leuV, were observed, and V-val was the most common subtype in all the three groups, followed by P-thrV and V-leuV. The distribution of the EBNA1 subtypes among EBVaGC, NPC and healthy donors was not significantly different (P >0.05). In addition, preferential linkages between EBNA1 subtypes and EBNA3C variants were found to exist. There was no evidence that particular EBNA1 subtypes are preferentially associated with EBVaGC or NPC in Northern China, suggesting that EBNA1 gene variations are geographically restricted rather than tumor-specific polymorphisms. [Copyright &y& Elsevier]

• The protein levels of ULK1 were down-regulated in EBV-associated gastric carcinoma cell lines compared with EBV-negative gastric carcinoma. • EBV-miR-BART2-3p targets the 3'UTR of ULK1 and regulates its expression. • miR-BART2-3p regulates autophagy and migration by targeting ULK1, but does not effect cell proliferation. ULK1 (Unc-51-like kinase 1) is an evolutionarily conserved serine/threonine kinase that plays a central role in the regulation of autophagy. ULK1 is associated with prognosis for metastasis and survival in several tumors. However, its relationship with Epstein-Barr virus (EBV) has not been studied. We found that the expression of ULK1 in EBV-associated gastric cancer cells was lower than that in EBV-negative gastric cancer cells. Further, a luciferase reporter gene assay showed that miR-BART2-3p directly targets ULK1. EBV-miR-BART2-3p attenuated endogenous protein expression levels of some autophagy-related genes. MiR-BART2-3p could thus be involved in the regulation of autophagy. Most important, our research indicates that miR-BART2-3p targets ULK1, resulting in downregulation of epithelial-mesenchymal transformation (EMT) -associated marker proteins and reducing EMT and cell migration. Our study shows that modulation of ULK1 is the likely mechanism by which miR-BART2-3p participates in the regulation of autophagy and cancer cell migration. [ABSTRACT FROM AUTHOR]

• The m6A total RNA methylation level of the EBVnGC cell line (SGC7901) was higher than that in the EBVaGC cell line (GT38). • the m6A "writer" WTAP protein in EBVaGC cell lines was significantly lower than that in EBVnGC cell lines. • EBV-encoded small RNA1 (EBER1) could downregulate WTAP expression by activating the NF-κB signaling pathway. • WTAP could increase proliferation and inhibit migration in gastric carcinoma cell lines. Epstein-Barr virus-associated gastric carcinoma (EBVaGC) is characterized by the clonal growth of EBV-infected stomach epithelial cells. It has been reported that N6-methyladenosine (m6A) methylation can regulate the splicing, expression, decay and translation of mRNAs. Wilms' tumor 1-associating protein (WTAP) is an m6A "writer" with methyltransferase activity. An m6A RNA methylation quantification kit and immunofluorescence (IF) showed that the m6A total RNA methylation level of the Epstein-Barr virus-negative gastric carcinoma (EBVnGC) cell line (SGC7901) was higher than that in the EBVaGC cell line (GT38). To investigate the underlying mechanism of the downregulated expression of m6A RNA methylation, we analyzed the expression of WTAP. The results showed that the expression of WTAP protein in EBVaGC cell lines was significantly lower than that in EBVnGC cell lines according to western blotting and IF. Through plasmid overexpression and RNA interference technology, we further found that EBV-encoded small RNA1 (EBER1) could downregulate WTAP expression by activating the NF-κB signaling pathway. In addition, WTAP could increase proliferation and inhibit migration in gastric carcinoma cell lines. In summary, EBER1 of EBV potentially regulated WTAP by affecting the NF-κB signaling pathway and WTAP further affected cell proliferation and migration. [ABSTRACT FROM AUTHOR]

• The mRNA and protein levels of MAP9 were up-regulated in EBV-positive gastric carcinoma cell lines. • The positive rate of MAP9 expression in EBVaGC tissues was higher than that in EBV-negative gastric carcinoma tissues. • EBV may regulate MAP9 expression by modifying the methylation of MAP9 CpG islands through DNMT1. • By inhibiting MAP9 in GT38 and overexpressing MAP9 in AGS, we proved that MAP9 inhibited the growth and induced apoptosis of EBVaGC cells. By inhibiting MAP9 in GT38 and overexpressing MAP9 in AGS, MAP9 can inhibit. Epstein–Barr virus (EBV)-associated gastric carcinoma (GC) comprises approximately 9% of all cases of GC. EBV-associated GC (EBVaGC) has characteristic clinicopathological features for a favorable prognosis. Microtubule-associated protein 9 (MAP9) is a cell cycle-associated gene required for bipolar spindle assembly, mitosis progression, and cytokinesis. Nevertheless, to date, there have been no reports on MAP9 function in EBVaGC. In this study, we demonstrated that the mRNA and protein levels of MAP9 were up-regulated in EBV-positive gastric carcinoma cell lines. The positive rate of MAP9 expression in EBVaGC tissues was shown to be significantly higher than that in EBV-negative gastric carcinoma (EBVnGC) tissues. Additionally, the expression of MAP9 was partly increased in EBVnGC cell lines by interfering with DNA methyltransferase 1 (DNMT1) or treated with 5-aza-2′-deoxycytidine. Thus, EBV may regulate MAP9 expression by modifying the methylation of MAP9 CpG islands through DNMT1. By inhibiting the expression of MAP9 with small interfere sequence in the EBV-positive GC cell line GT38 and overexpressing MAP9 in the EBV-negative GC cell line AGS, we demonstrated that MAP9 inhibited the growth and induced apoptosis of EBVaGC cells significantly. In conclusion, our study demonstrated that EBV can up-regulate the expression of MAP9 in EBVaGC, and the methylation of MAP9 CpG islands influences this regulation. And MAP9 acts as a tumor suppressor in the development of EBVaGC. [ABSTRACT FROM AUTHOR]