Esra Dirimtekin, D. Alwyn Dart, Pinar Uysal-Onganer
Epstein–Barr virus (EBV), a ubiquitous human herpesvirus infecting over 90% of the adult population, is causally associated with a distinct molecular subtype of gastric cancer (GC). A key mechanism by which EBV influences tumour biology is the expression of viral microRNAs (miR/miRNA) encoded within the BamHI-A rightward transcript (BART) region, although inter-patient variability in EBV-miRNA expression and its molecular significance remain incompletely defined. In this study, small RNA sequencing was performed on 11 primary gastric tumour samples to characterise EBV-derived miRNA expression, followed by quantitative RT-PCR analysis in an extended cohort of 21 tumours for targeted validation. EBV-miRNAs were detected in a subset of tumours and showed marked inter-tumour heterogeneity in abundance. EBV-miRNA-positive tumours were dominated by a conserved set of BART miRNAs, including miR-BART19-3p, miR-BART1-5p, miR-BART10-3p, miR-BART6-3p, miR-BART13-5p, and miR-BART22. These BART miRNAs displayed correlated expression patterns, characterised by concurrent elevation of multiple viral miRNA species within the same tumour samples. To link viral miRNA expression with host molecular responses, in silico virus–host interaction analysis was conducted using ViRBase to prioritise host genes associated with the detected BART miRNAs. PTEN, BCL2L11, FOXO3, and CDKN1A were identified as high-confidence targets and selected for experimental assessment. RT-qPCR analysis demonstrated differential expression of these host genes across tumours stratified by EBV BART miRNA abundance. Together, these findings identify a consistent BART miRNA pattern within this cohort. This study provides patient-level molecular evidence linking EBV-miRNA regulatory output to host gene expression states in GC.