Stearoyl-CoA desaturase 1 is targeted by EBV-encoded miR-BART20-5p and regulates cell autophagy, proliferation, and migration in EBV-associated gastric cancer.

Epstein-Barr virus (EBV) is the first human oncogenic virus known to express microRNAs (miRNAs), which are closely associated with the development of various tumors, including nasopharyngeal and gastric cancers. Stearoyl-CoA Desaturase 1 (SCD1) is a key enzyme in fatty acid synthesis, highly expressed in numerous tumors, promoting tumor growth and metastasis, making it a potential therapeutic target. In this study, we found that SCD1 expression in EBV-associated gastric cancer (EBVaGC) was significantly lower than in EBV-negative gastric cancer (EBVnGC) at both cellular and tissue levels. In addition, EBV-miR-BART20-5p targets the 3'-UTR of SCD1, downregulating its expression. Moreover, overexpression of SCD1 in EBVaGC cells promoted cell migration and proliferation while inhibiting autophagy. These results suggest that EBV-encoded miRNA-BART20-5p may contribute to EBVaGC progression by targeting SCD1.

METTL3 regulates M6A methylation-modified EBV-pri-miR-BART3-3p to promote NK/T cell lymphoma growth.

OBJECTIVE: N6-methyladenosine (M6A) is the most prevalent epigenetic alteration. Methyltransferase-like 3 (METTL3) is a key player in the control of M6A modification. Methyltransferase promote the processing of mature miRNA in an M6A-dependent manner, thereby participating in disease occurrence and development. However, the regulatory mechanism of M6A in NK/T cell lymphoma (NKTCL) remains unclear. PATIENTS AND METHODS: We determined the expression of METTL3 and its correlation with clinicopathological features using qRT-PCR and immunohistochemistry. We evaluated the effects of METTL3 on NKTCL cells using dot blot assay, CCK8 assay and subcutaneous xenograft experiment. We then applied M6A sequencing combined with gene expression omnibus data to screen candidate targets of METTL3. Finally, we investigated the regulatory mechanism of METTL3 in NKTCL by methylated RNA immunoprecipitation and RNA immunoprecipitation (RIP) assays. RESULTS: We demonstrated that METTL3 was highly expressed in NKTCL cells and tissues and indicated poor prognosis. The METTL3 expression was associated with NKTCL survival. Functionally, METTL3 promoted the proliferation capability of NKTCL cells in vitro and in vivo. Furthermore, EBV-miR-BART3-3p was identified as the downstream effector of METTL3, and silencing EBV-miR-BART3-3p inhibited the proliferation of NKTCL. Finally, we confirmed that PLCG2 as a target gene of EBVmiR-BART3-3p by relative assays. CONCLUSIONS: We identified that METTL3 is significantly up-regulated in NKTCL and promotes NKTCL development. M6A modification contributes to the progression of NKTCL via the METTL3/EBV-miR-BART3-3p/PLCG2 axis. Our study is the first to report that M6A methylation has a critical role in NKTCL oncogenesis, and could be a potential target for NKTCL treatment.

BPIFB1, Serving as a Downstream Effector of EBV-miR-BART4, Blocks Immune Escape of Nasopharyngeal Carcinoma via Inhibiting PD-L1 Expression.

Nasopharyngeal carcinoma (NPC) is one of the most common tumors of head and neck in the Southeast Asia. PD-L1-dependent immune escape plays a critical role involved in NPC development. BPIFB1 has previously reported to take tumor-suppressive actions on NPC cell proliferation and migration. Nonetheless, the function of BPIFB1 in immune escape remains largely elusive. Expression pattern on mRNA and protein levels of target genes in NPC patients' samples and cell lines were examined by qRT-PCR, western blot, and immunohistochemistry staining, respectively. The assessment of CD8+ T-cell apoptosis and expression was determined by flow cytometry. Molecular interactions were verified using chromatin immunoprecipitation (ChIP) and luciferase reporter assay. BPIFB1 was downregulated in NPC tumor tissues, exhibiting a negative correlation of PD-L1. Overexpression of BPIFB1 significantly inhibited the expression of PD-L1, suppressing the apoptosis and enhancing the expression of CD8+ T cells. Mechanistically, BPIFB1 was found to repress the expression of STAT1, which was identified to be an upstream activator of PD-L1. Furthermore, the EBV-encoded miR-BART4 overexpressed in NPC cells could directly target and inhibit BPIFB1. This study provided a comprehensive understanding of the molecular mechanism for the upstream and downstream pathway of BPIFB1 related with immune escape, indicating a novel approach for the treatment of NPC.

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

Background: 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. Methods: 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. Results: 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. Conclusions: 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.

Epstein-Barr virus-encoded miR-BART11-3p modulates the DUSP6-MAPK axis to promote gastric cancer cell proliferation and metastasis.

Epstein-Barr virus (EBV)-encoded miRNAs within the BamHI-A rightward transcript (BART) region are abundantly expressed in EBV-associated gastric cancer (EBVaGC), suggesting that they play roles in tumorigenesis. However, how these viral miRNAs contribute to the development of EBVaGC remains largely obscure. In this study, we found that EBV-encoded miR-BART11-3p targets 3' -UTR of dual-specificity phosphatase 6 (DUSP6) mRNA to upregulate ERK phosphorylation and downregulate JNK and p38 phosphorylation. By doing so, miR-BART11-3p promotes gastric cancer (GC) cell proliferation, migration, and invasion in vitro, and facilitates tumor growth in vivo. Restoration of DUSP6 expression reverses the tumor-promoting activity of miR-BART11-3p in AGS GC cells. Consistently, knockdown of DUSP6 ablates the antitumor effects of miR-BART11-3p inhibitors in EBV-positive GC cells. Furthermore, blocking ERK phosphorylation with trametinib inhibited the proliferation, migration, and invasion of miR-BART11-3p-expressing AGS cells. Administration of a miR-BART11-3p antagomir reduced the growth of EBV-positive xenograft tumors. Together, these findings reveal a novel mechanism by which EBV dysregulates MAPK pathways through an EBV-encoded microRNA to promote the development and progression of EBVaGC, which may be harnessed to develop new therapeutics to treat EBVaGC. IMPORTANCE The Epstein-Barr virus (EBV) is the first human tumor virus found to encode miRNAs, which within the BART region have been detected abundantly in EBV-associated gastric cancer (EBVaGC) and play various roles in promoting tumorigenesis. In our study, we observed that EBV-miR-BART11-3p promotes cell proliferation and induces migration and invasion in GC. Interestingly, we showed that miR-BART11-3p upregulates p-ERK and downregulates p-JNK and p-p38 by directly targeting 3'-UTR of dual-specificity phosphatase 6 (DUSP6). Restoration of DUSP6 rescues the effects generated by miR-BART11-3p in GC cells, and blocking ERK phosphorylation with Trametinib augments JNK and p38 phosphorylation and inhibits the effects of miR-BART11-3p-expressing AGS cells, suggesting that miR-BART11-3p promotes cell proliferation, migration, and invasion by modulating DUSP6-MAPK axis in EBVaGC. The findings presented in this study provide new mechanisms into the tumorigenesis in EBVaGC and new avenues for the development of therapeutic strategies to combat EBVaGC targeting miR-BART11-3p or phospho-ERK.

Epstein-Barr virus microRNA miR-BART2-5p accelerates nasopharyngeal carcinoma metastasis by suppressing RNase â ¢ endonuclease DICER1.

The development and progression of nasopharyngeal carcinoma (NPC) is closely associated with Epstein-Barr virus (EBV) infection. NPC is usually asymptomatic until it spreads to other sites, and more than 70% of cases are classified as locally advanced disease at diagnosis. EBV-positive nasopharyngeal cancer tissues express only limited viral latent proteins, but express high levels of the EBV-encoded BamHI-A rightward transcript (BART) miRNA molecules. Here, we report that EBV-miRNA-BART2-5p (BART2-5p) promotes NPC cell invasion and metastasis in vivo and in vitro but has no effect on NPC cell proliferation and apoptosis. In addition, BART2-5p altered the mRNA and miRNA expression profiles of NPC cells. The development of human tumors has been reported to be associated with altered miRNAs expression, and overall miRNAs expression is reduced in many types of tumors. We found that BART2-5p downregulated the expression of several miRNAs that could exert oncogenic functions. Mechanistically, BART2-5p directly targets the RNase III endonuclease DICER1, inhibiting its function of cleaving double-stranded stem-loop RNA into short double-stranded RNA, which in turn causes altered expression of a series of key epithelial-mesenchymal transition molecules, and reverting DICER1 expression can rescue this phenotype. Furthermore, analysis from clinical samples showed a negative correlation between BART2-5p and DICER1 expression. According to our study, high expression of BART2-5p in tissues and plasma of patients with NPC is associated with poor prognosis. Our results suggest that, BART2-5p can accelerate NPC metastasis through modulating miRNA profiles which are mediated by DICER1, implying a novel role of EBV miRNAs in the pathogenesis of NPC.

Effects of Co-Culture EBV-miR-BART1-3p on Proliferation and Invasion of Gastric Cancer Cells Based on Exosomes.

AIM: EBV encodes at least 44 miRNAs involved in immune regulation and disease progression. Exosomes can be used as carriers of EBV-miRNA-BART intercellular transmission and affect the biological behavior of cells. We characterized exosomes and established a co-culture experiment of exosomes to explore the mechanism of miR-BART1-3p transmission through the exosome pathway and its influence on tumor cell proliferation and invasion. MATERIALS AND METHODS: Exosomes of EBV-positive and EBV-negative gastric cancer cells were characterized by transmission electron microscopy. NanoSight and Western blotting, and miRNA expression profiles in exosomes were sequenced with high throughput. Exosomes with high or low expression of miR-BART1-3p were co-cultured with AGS cells to study the effects on proliferation, invasion, and migration of gastric cancer cells. The target genes of EBV-miR-BART1-3p were screened and predicted by PITA, miRanda, RNAhybrid, virBase, and DIANA-TarBase v.8 databases, and the expression of the target genes after co-culture was detected by qPCR. RESULTS: The exosomes secreted by EBV-positive and negative gastric cancer cells range in diameter from 30 nm to 150 nm and express the exosomal signature proteins CD9 and CD63. Small RNA sequencing showed that exosomes expressed some human miRNAs, among which hsa-miR-23b-3p, hsa-miR-320a-3p, and hsa-miR-4521 were highly expressed in AGS-exo; hsa-miR-21-5p, hsa-miR-148a-3p, and hsa-miR-7-5p were highly expressed in SNU-719-exo. All EBV miRNAs were expressed in SNU-719 cells and their exosomes, among which EBV-miR-BART1-5p, EBV-miR-BART22, and EBV-miR-BART16 were the highest in SNU-719 cells; EBV-miR-BART1-5p, EBV-miR-BART10-3p, and EBV-miR-BART16 were the highest in SNU-719-exo. After miR-BART1-3p silencing in gastric cancer cells, the proliferation, healing, migration, and invasion of tumor cells were significantly improved. Laser confocal microscopy showed that exosomes could carry miRNA into recipient cells. After co-culture with miR-BART1-3p silenced exosomes, the proliferation, healing, migration, and invasion of gastric cancer cells were significantly improved. The target gene of miR-BART1-3p was FAM168A, MACC1, CPEB3, ANKRD28, and USP37 after screening by a targeted database. CPEB3 was not expressed in all exosome co-cultured cells, while ANKRD28, USP37, MACC1, and FAM168A were all expressed to varying degrees. USP37 and MACC1 were down-regulated after up-regulation of miR-BART1-3p, which may be the key target genes for miR-BART1-3p to regulate the proliferation of gastric cancer cells through exosomes. CONCLUSIONS: miR-BART1-3p can affect the growth of tumor cells through the exosome pathway. The proliferation, healing, migration, and invasion of gastric cancer cells were significantly improved after co-culture with exosomes of miR-BART1-3p silenced expression. USP37 and MACC1 may be potential target genes of miR-BART1-3p in regulating cell proliferation.

Downregulation of MUS81 expression inhibits cell migration and maintains EBV latent infection through miR-BART9-5p in EBV-associated gastric cancer.

Epstein-Barr virus (EBV) infection is associated with the occurrence and development of gastric cancer (GC). Methyl methanesulfonate and ultraviolet-sensitive gene 81 (MUS81) is the catalytic component of a structure-specific endonuclease and plays an important role in chromosomal stability. However, the link between EBV infection and MUS81 remains unclear. In the present study, we found that MUS81 expression was much lower in EBV-associated GC cells than in EBV-negative GC. MUS81 acts as an oncogene in GC by inducing the cell migration and proliferation. Western blot and luciferase reporter assays revealed that miR-BART9-5p directly targeted MUS81 and downregulated its expression. Additionally, overexpression of MUS81 in EBV-positive GC cells inhibited the expression of EBV nuclear antigen 1 (EBNA1). EBNA1 is critical for the pathogenesis of EBV-associated tumors and the maintenance of a stable copy number of the viral genomes. Altogether, these results indicated that the lowering MUS81 expression might be a mechanism by EBV to maintain its latent infection.

Quantitative analysis of Epstein-Barr virus DNA in plasma and stomach biopsies of patients with gastric cancer.

Epstein-Barr virus (EBV) associated gastric carcinoma (EBVaGC) is a subtype of gastric cancer with distinct histological and molecular features. The study aimed to assess the EBV DNA copy number and the prevalence of EBVaGC in gastric cancer samples taken from Iranian patients. The next aim was to assess whether the DNA and microRNAs EBV are present in plasma. EBV load was analyzed in 68 gastric cancer biopsies and compared with the results of EBV-encoded small RNA in situ hybridization (EBER-ISH) test in these patients. After the detection of 6 EBV miRNAs in gastric tissue by stem-loop RT-PCR, plasma samples were evaluated for the viral load and EBV miRNAs. Four gastric cancer cases were EBER -ISH positive (5.8%), with a significantly higher viral load than the remaining cases, 47,781 vs. 1909 copies/µg of tissue DNA. Here, was also found a significant difference in plasma EBV load between EBER-positive and EBER-negative cases. Although EBV miRNAs were detectable in all the EBER-positive tumors, the test did not detect any of these miRNAs among the plasma samples tested. Our data indicate that the prevalence of EBVaGC among Iranian patients with gastric cancer is lower than the global prevalence and although none of the EBV miRNAs were detected in plasma, evaluation of EBV microRNAs in tumor tissue, especially miR-BART7-3p, may constitute useful biomarkers for diagnosis of EBVaGC.

Exosomes loaded with circPARD3 promotes EBV-miR-BART4-induced stemness and cisplatin resistance in nasopharyngeal carcinoma side population cells through the miR-579-3p/SIRT1/SSRP1 axis.

OBJECTIVE: To explore the effects of exosomes loaded with circular RNA PARD3 on EBV-miR-BART4-induced stemness and resistance of cisplatin in nasopharyngeal carcinoma side population (NPC-SP) cells through the miR-579-3p/SIRT1/SSRP1 axis. METHODS: Sixty-five cancer tissues and 65 noncancerous tissues were collected from NPC patients or patients with rhinitis. The expressions of circPARD3, miR-579-3p, SIRT1, and SSRP1 were detected by qRT-PCR, western blot, or immunohistochemistry. In vivo tumor formation assay was performed in nude mice. Immunofluorescence and qRT-PCR were conducted for the determination of CD44 and CD133 expressions, and flow cytometry combined with Hoechst 33,342 dye efflux for identifying SP cells, CCK-8 and EdU assays for cell proliferation, and Transwell assay for migration and invasion. RESULTS: CircPARD3, SIRT1, and SSRP1 were upregulated while miR-579-3p was downregulated in NPC tissues and cells. CircPARD3 was positively correlated with the expressions of SIRT1 and SSRP1, and miR-579-3p was negatively correlated with circPARD3, SIRT1, and SSRP1. Exosomes loaded with circPARD3 promoted EBV-miR-BART4-induced stemness and cisplatin resistance in NPC-SP cells, while miR-579-3p reversed the effect of exosomal circPARD3 on EBV-miR-BART4-induced stemness and cisplatin resistance in NPC-SP cells. Additionally, miR-579-3p suppressed EBV-miR-BART4-induced stemness and cisplatin resistance in NPC-SP cells by regulating SIRT1. SIRT1 upregulated SSRP1 expression by catalyzing H3K4 methylation and down-regulation of SSRP1 reversed the effect of SIRT1 on EBV-miR-BART4-induced stemness and cisplatin resistance in NPC-SP cells. CONCLUSION: Exosomes loaded with circPARD3 promoted EBV-miR-BART4-induced stemness and cisplatin resistance in NPC-SP cells through the miR-579-3p/SIRT1/SSRP1 axis. Graphical Headlights • EBV-miR-BART4 induces the stemness and resistance of NPC-SP cells. • CircPARD3 regulates SIRT1 by miR-579-3p. • SIRT1 regulates SSRP1 expression by histone methylation. • Exosomes loaded with circPARD3 promotes EBV-miR-BART4-induced NPC-SP cell stemness and resistance by the miR-579-3p/SIRT1/SSRP1 axis.

CircEAF2 counteracts Epstein-Barr virus-positive diffuse large B-cell lymphoma progression via miR-BART19-3p/APC/ß-catenin axis.

BACKGROUND: Epstein-Barr virus (EBV) represents an important pathogenic factor of lymphoma and is significantly associated with poor clinical outcome of diffuse large B-cell lymphoma (DLBCL). Circular RNAs (circRNAs) play an essential role in lymphoma progression. However, the underlying mechanism of circRNA on DLBCL progression related to EBV remains largely unknown. METHODS: CircRNA was screened by high-throughput sequencing in tumor samples of 12 patients with DLBCL according to EBV infection status. Expression of circEAF2, as well as the relationship with clinical characteristics and prognosis, were further analyzed in tumor samples of 100 DLBCL patients using quantitative real-time PCR. Gain- and loss-of-function experiments were conducted to investigate the biological functions of circEAF2 both in vitro and in vivo. The underlying mechanism of circRNA on DLBCL progression were further determined by RNA sequencing, RNA pull down assay, dual-luciferase reporter assay, rescue experiments and western blotting. RESULTS: We identified a novel circRNA circEAF2, which was downregulated in EBV + DLBCL and negatively correlated with EBV infection and DLBCL progression. In EBV-positive B lymphoma cells, circEAF2 overexpression induced lymphoma cell apoptosis and sensitized lymphoma cells to epirubicin. As mechanism of action, circEAF2 specifically targeted EBV-encoded miR-BART19-3p, upregulated APC, and suppressed downstream ß-catenin expression, resulting in inactivation of Wnt signaling pathway and inhibition of EBV + DLBCL cell proliferation. In EBV-positive B-lymphoma murine models, xenografted tumors with circEAF2 overexpression presented decreased Ki-67 positivity, increased cell apoptosis and retarded tumor growth. CONCLUSIONS: CircEAF2 counteracted EBV + DLBCL progression via miR-BART19-3p/APC/ß-catenin axis, referring circEAF2 as a potential prognostic biomarker. Therapeutic targeting EBV-encoded miRNA may be a promising strategy in treating EBV-associated lymphoid malignancies.

MiR-BART2-3p targets Unc-51-like kinase 1 and inhibits cell autophagy and migration in Epstein-Barr virus-associated gastric cancer.

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.

Epstein-Barr virus (EBV) encoded microRNA BART8-3p drives radioresistance-associated metastasis in nasopharyngeal carcinoma.

Radiotherapy plays an important role in the treatment of nasopharyngeal carcinoma (NPC), however, 20% of patients with NPC exhibit unusual radioresistance. Patients with radioresistance are at risk of recurrence, so it is imperative to explore the mechanism of resistance to radiotherapy. In the past, studies on the mechanism of radioresistance have been restricted to DNA damage and related cell cycle remodeling or apoptosis. So far, no studies have explored the relationship between radioresistance and metastasis. Through the analysis of clinical samples, we observed that the metastasis rate of recurrent NPC was much higher than that of primary patients. In vitro and in vivo experiments showed that NPC cells with acquired radioresistance exhibited a stronger ability for invasion and metastasis. Mechanistically, we found that the Epstein-Barr virus (EBV)-encoded miRNA BART8-3p was increased in patients with NPC, and its expression was positively correlated with adverse prognostic factors, such as radioresistance. Besides this, miR-BART8-3p promoted the epithelial-mesenchymal transition, invasion, and metastasis of radioresistant NPC cells by targeting and inhibiting their PAG1 host gene. These findings suggested a novel role for EBV-miR-BART8-3p in promoting NPC radioresistance-associated metastasis and highlighted its potential value as a prognostic indicator or therapeutic target.

As Evidence-Based Tumorigenic Role of Epstein-Barr Virus miR-BART1-3p in Neurological Tumors.

INTRODUCTION: Central nervous system tumors are a diverse group of tumors that account for 2% of all adult cancers and 17% of childhood malignancies. Several internal and external risk factors are involved in the development of this cancer such as viral infections. The aim of this study was to the determination of the EBV infection frequency and the expression level of miR-122 and miR-BART in CNS tumors samples. METHODS: One hundred and thirty-eight  fresh tissue sample (106 case and 32 control) was collected from CNS specimens. The presence of Epstein-Barr virus (EBV) DNA was examined by PCR assay and the expression level of miR-122 and miR-BART were evaluated by using real-time PCR assay in CNS tissue samples. RESULTS: EBV DNA was detected in 17% (18 of 106) of tumors tissue samples and 6.4% (2 of 32) of control samples. according to results, there was a significant relationship between the presence of EBV-DNA with CNS tumors. Additionally, the expression level of miR-122 was significantly downregulated in the EBV-positive sample compared to that of the EBV-negative sample. Also, the level of EBV-BART1-3p expression was significantly higher in EBV-positive tumors samples than EBV-positive normal samples. CONCLUSION: The results of this study suggest that the EBV could change the condition of cancer cells by altering the expression of miR-122 and EBV-BART1-3p and maybe contribute to the development of cancer cells. However, the role of viral infections in CNS cancer requires further studies. 
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Effects of Dicer1 targeted by EBV-miR-BART6-5p on biological properties and radiosensitivity of nasopharyngeal carcinoma.

OBJECTIVE: To discuss the effects of Epstein-Barr virus (EBV)-encoded BamHI A rightward transcript (BART) microRNA (miR-BART6-5p) by targeting Dicer1 on biological properties and radiosensitivity of nasopharyngeal carcinoma (NPC). METHODS: NPC patients (n = 96) treated with radiotherapy were collected from Jan 2010 to Jan 2011. Real-time quantitative PCR (qRT-PCR) and western blot were carried out to measure the expression of miR-BART6-5p and Dicer1. Dual luciferase reporter gene assay verified that miR-BART6-5p targeted Dicer1. CCK8, wound-healing, Transwell and Annexin-FITC/PI were employed to evaluate the effects of Dicer1 mediated by miR-BART6-5p on biological characteristics of NPC cells. The radiosensitivity of miR-BART6-5p targeting Dicer1 was assessed in vitro and in vivo. RESULTS: Increased miR-BART6-5p and decreased Dicer1 were discovered in NPC patients, displaying a close association with T-stage, clinical stage, as well as Pre-DNA of NPC. While elevated Dicer1 and miR-BART6-5p down-regulation in NPC patients were found after effective radiotherapy. Both miR-BART6-5p and Dicer1 were prognostic factors of NPC. Down-regulation of miR-BART6-5p could enhance Dicer1 expression and inhibit NPC cell proliferation, invasion and migration with promoted apoptosis. Clone formation assay also showed miR-BART6-5p down-regulation reduced planting efficiency (PE), which further decreased with the increased dose of irradiation. Injection with miR-BART6-5p inhibitors in nude mice after 6-Gy irradiation contributed to the overexpression of Dicer1 and the inhibition of tumor growth. CONCLUSIONS: EBV-miR-BART6-5p may target Dicer1 to facilitate proliferation and metastasis of NPC cells and suppress apoptosis, thus being a new target for NPC therapy.

EBV-miR-BART12 accelerates migration and invasion in EBV-associated cancer cells by targeting tubulin polymerization-promoting protein 1.

Epstein-Barr virus (EBV) infection leads to cancers with an epithelial origin, such as nasopharyngeal cancer and gastric cancer, as well as multiple blood cell-based malignant tumors, such as lymphoma. Interestingly, EBV is also the first virus found to carry genes encoding miRNAs. EBV encodes 25 types of pre-miRNAs which are finally processed into 44 mature miRNAs. Most EBV-encoded miRNAs were found to be involved in the occurrence and development of EBV-related tumors. However, the function of EBV-miR-BART12 remains unclear. The findings of the current study revealed that EBV-miR-BART12 binds to the 3'UTR region of Tubulin Polymerization-Promoting Protein 1 (TPPP1) mRNA and downregulates TPPP1, thereby promoting the invasion and migration of EBV-related cancers, such as nasopharyngeal cancer and gastric cancer. The mechanism underlying this process was found to be the inhibition of TPPP1 by EBV-miRNA-BART12, which, in turn, inhibits the acetylation of α-tubulin, and promotes the dynamic assembly of microtubules, remodels the cytoskeleton, and enhances the acetylation of ß-catenin. ß-catenin activates epithelial to mesenchymal transition (EMT). These two processes synergistically promote the invasion and metastasis of tumor cells. To the best of our knowledge, this is the first study to reveal the role of EBV-miRNA-BART12 in the development of EBV-related tumors as well as the mechanism underlying this process, and suggests potential targets and strategies for the treatment of EBV-related tumors.

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

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Epstein-Barr virus-encoded miR-BART11 promotes tumor-associated macrophage-induced epithelial-mesenchymal transition via targeting FOXP1 in gastric cancer.

Gastric carcinoma (GC) is an Epstein-Barr virus (EBV)-associated malignancy characterized by early metastasis. Unlike that of cellular micro(mi)RNAs, the role of viral miRNAs in epithelial-mesenchymal transition (EMT) and metastasis in cancers has not been fully investigated. In this study, we elucidated the involvement of miR-BART11, an EBV-encoded viral miRNA, in the EMT and metastasis of GC cells. EBV-miR-BART11 upregulation can lead to downregulation of forkhead box protein P1 (FOXP1) in both tissues and cell lines of gastric carcinoma. Downregulation of FOXP1 might trigger the secretion of interleukin 1ß (IL-1ß), IL-6, and 1L-10 in cancer cells, resulting in poor survival of GC patients. We found that the observed EMT phenotypes resulted from the EBV-miR-BART11 overexpression-induced FOXP1 downregulation, which impacted the expression of the EMT-transcription factors E-cadherin and snail. We further demonstrated that conditioned medium-derived tumor-associated macrophages (TAMs) promoted phenotypic changes and expression of EMT-related molecules in GC cells. Additionally, EMT changes were significantly promoted in GC cells cultured in conditioned medium from TAMs infected with EBV-miR-BART11-containing lentivirus. On the contrary, GC cells cultured in conditioned medium from TAMs infected with FOXP1-carrying lentivirus showed little or no EMT change. Taken together, our results suggest that EBV-encoded viral miRNA BART11 downregulates the FOXP1 transcription factor, and promotes EMT by directly influencing gastric tumor cells or indirectly affecting the tumor microenvironment, which might, in turn, accelerate cancer invasion and metastasis, thereby affecting the survival and prognosis of patients.

Epstein-Barr virus-encoded miR-BART5-5p upregulates PD-L1 through PIAS3/pSTAT3 modulation, worsening clinical outcomes of PD-L1-positive gastric carcinomas.

BACKGROUND: Epstein-Barr virus (EBV) is etiologically associated with ~ 10% of all gastric carcinomas. However, the molecular mechanisms and roles of EBV miRNAs in gastric carcinoma oncogenesis are yet to be elucidated. METHODS: MicroRNA microarray and TaqMan quantitative real-time RT-PCR were conducted. RT-PCR and luciferase reporter assay for PIAS3, western blotting for 20 proteins, immunofluorescence for STAT3, transfection with miRBART5-5p-plasmid, STAT3-plasmid, miRBART5-5p mimic, or PIAS3-siRNA, and in vitro assays for biological effects of PD-L1 were implemented. In situ hybridization for EBV-encoded small RNAs and immunohistochemistry were performed on gastric carcinoma tissues. RESULTS: Transfecting miR-BART5-5p into EBV(-) gastric carcinoma cell lines caused a decrease in PIAS3 3'-UTR reporter activity, PIAS3 downregulation, and subsequent STAT3 activation followed by PIAS3/pSTAT3-dependent PD-L1 upregulation. Interestingly, due to PD-L1 knockdown, apoptosis was increased, while the rate of cell proliferation, invasion capacity, and migration were decreased in miR-BART5-5p-transfected cells. In EBV(+) gastric carcinoma cells, anti-miR-BART5-5p reduced PD-L1 levels through PIAS3/pSTAT3 control. Among 103 patients with EBV-associated gastric carcinomas, overall survival was significantly shortened for those with PD-L1(+) tumors compared to those with PD-L1(-) tumors (P = 0.049). CONCLUSIONS: Our findings imply that miR-BART5-5p directly targets PIAS3 and augments PD-L1 through miR-BART5/PIAS3/pSTAT3/PD-L1 axis control. This contributes to antiapoptosis, tumor cell proliferation, invasion and migration, as well as immune escape, furthering gastric carcinoma progression and worsening the clinical outcome, especially in the PD-L1(+) group of patients with EBV-associated gastric carcinomas. miR-BART5-5p may, therefore, be amenable to PD-1/PD-L1 immune checkpoint inhibitor therapy.

MiR-BART1-5p targets core 2ß-1,6-acetylglucosaminyltransferase GCNT3 to inhibit cell proliferation and migration in EBV-associated gastric cancer.

GCNT3 (core 2ß-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.