Profiling of Virus-Encoded MicroRNAs in Epstein-Barr Virus-Associated Gastric Carcinoma and Their Roles in Gastric Carcinogenesis

Abstract

Epstein-Barr virus (EBV) is one of the major oncogenic viruses and is found in nearly 10% of gastric carcinomas. EBV is known to encode its own microRNAs (miRNAs); however, their roles have not been fully investigated. The present report is the largest series to comprehensively profile the expression of 44 known EBV miRNAs in tissue samples from patients with EBV-associated gastric carcinoma. Several miRNAs were highly expressed in EBV-associated gastric carcinoma, and in silico analysis revealed that the target genes of these EBV miRNAs had functions associated with cancer-related pathways, especially the regulation of apoptosis. Apoptosis was reduced in EBV-associated gastric carcinoma tissue samples, and gastric carcinoma cell lines infected with EBV exhibited downregulation of the proapoptotic protein Bid (the BH3-interacting domain death agonist), a member of the Bcl-2 family. The luciferase activity of the reporter vector containing the 3= untranslated region of BID was inhibited by an ebv-miR-BART4-5p mimic in gastric cancer cell lines. Transfection of an ebvmiR-BART4-5p mimic reduced Bid expression in EBV-negative cell lines, leading to reduced apoptosis under serum deprivation. The inhibition of ebv-miR-BART4-5p expression was associated with partial recovery of Bid levels in EBV-positive cell lines. The results demonstrated the antiapoptotic role of EBV miRNA via regulation of Bid expression in EBV-associated gastric carcinoma. These findings provide novel insights in the roles of EBV miRNAs in gastric carcinogenesis, which would be a potential therapeutic target. IMPORTANCEThis report is the largest series to comprehensively profile the expression of 44 known EBV miRNAs in clinical samples from EBV-associated gastric carcinoma patients. Of the EBV miRNAs, ebv-miR-BART4-5p plays an important role in gastric carcinogenesis via regulation of apoptosis. E pstein-Barr virus (EBV) is one of the major oncogenic viruses inducing various kinds of malignancies in humans. The virus originally was isolated from Burkitt's lymphoma and is associated with other hematopoietic tumors, including Hodgkin's lymphoma, extranodal nasal-type NK/T-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), and lymphoproliferative disorders in immunosuppressed patients, as well as some epithelial malignancies, such as nasopharyngeal carcinoma (NPC) and gastric carcinoma (1-3). Nearly 10% of all gastric carcinomas are associated with EBV infection and form a distinct subtype of disease, exhibiting characteristic clinicopathological features (4).Several studies have investigated the roles played by EBV in carcinogenesis and have sought to show that EBV gene transcripts, including EBV nuclear antigen 2 (EBNA2) or latent membrane protein 1 (LMP1), disturb signal transduction in host cells, causing immortalization or transformation (5). Although these studies have shed light on the oncogenic properties of EBV, many unsolved problems remain.Recently, a novel class of small, noncoding RNA molecules, termed microRNAs (miRNAs), has ...

EBV BART MicroRNAs Target Multiple Pro-apoptotic Cellular Genes to Promote Epithelial Cell Survival

“…These include BID, a proposed target for miR-BART4 [21]; PUMA, a proposed target for miR-BART5 [22]; PTEN, a proposed target for miR-BART7 [24]; E-Cadherin (E-CAD), a proposed target for miR-BART9 [43]; and finally, EBF1, a proposed target for miR-BART11 [44]. All of these miRNAs, except miR-BART4, were expressed at physiological levels in the AGS transductants (Fig 1), so the lack of a detectable anti-apoptotic phenotype was unexpected.…”

Outlooks on Epstein-Barr virus associated gastric cancer

“…EBV was the first virus in which miRNAs were discovered [40]. It contains approximately 25 miRNA precursors and 44 mature miRNAs, which are divided into two major clusters: (a) BART and (b) BHRF-1 [41]. The genes targeted by EBV miRNAs are associated with oncogenesis, cell adhesion, signal transduction and apoptosis, all of which contribute to carcinogenesis [41].…”

“…It contains approximately 25 miRNA precursors and 44 mature miRNAs, which are divided into two major clusters: (a) BART and (b) BHRF-1 [41]. The genes targeted by EBV miRNAs are associated with oncogenesis, cell adhesion, signal transduction and apoptosis, all of which contribute to carcinogenesis [41]. For instance, BART reduces the expression of Bid, which is an apoptotic molecule [40].…”

Dissecting the regulation of EBV's BART miRNAs in carcinomas

“…The BART miRNAs foster EBV’s life-cycle by inhibiting apoptosis and immune responses to infected B-cells (Albanese et al, 2016; Lin et al, 2015; Tagawa et al, 2016; Vereide et al, 2014). The BART miRNAs also likely promote epithelial cell survival by targeting multiple pro-apoptotic cellular genes that could contribute to the EBV-mediated epithelial carcinogenesis (Cai et al, 2015; Kanda et al, 2015; Kang et al, 2015; Marquitz et al, 2014, 2012; Shinozaki-Ushiku et al, 2015). Most of EBV’s miRNAs are processed from two adjacent regions separated by one copy of oriLyt in the BART locus.…”