Next‐generation sequencing of miRNAs in clinical samples of Epstein–Barr virus‐associated B‐cell lymphomas

Epstein–Barr virus (EBV) encodes 49 microRNAs (miRNAs) in the BART and BHRF1 regions of its genome. Although expression profiles of EBV‐encoded miRNAs have been reported for EBV‐positive cell lines and nasopharyngeal carcinoma, to date there is little information about total miRNA expression, including cellular and viral miRNAs, in the primary tumors of EBV‐associated B‐lymphoproliferative disorders. In this study, next‐generation sequencing and quantitative real‐time reverse transcription‐PCR were used to determine the expression profiles of miRNAs in EBV‐infected cell lines and EBV‐associated B‐cell lymphomas, including AIDS‐related diffuse large B‐cell lymphoma (DLBCL), pyothorax‐associated lymphoma, methotrexate‐associated lymphoproliferative disorder, EBV‐positive DLBCL of the elderly, and Hodgkin lymphoma. Next‐generation sequencing revealed that EBV‐encoded miRNAs accounted for up to 34% of total annotated miRNAs in these cases. Expression of three miR‐BHRF1s was significantly higher in AIDS‐related DLBCL and pyothorax‐associated lymphoma compared with methotrexate‐associated lymphoproliferative disorder and EBV‐positive DLBCL of the elderly, suggesting the association of miR‐BHRF1s expression with latency III EBV infection. Heat map/clustering analysis of expression of all miRNAs, including cellular and EBV miRNAs, by next‐generation sequencing demonstrated that each EBV tumor, except methotrexate‐associated lymphoproliferative disorder, formed an isolated cluster. Principal component analysis based on the EBV‐encoded miRNA expression showed that each EBV tumor formed a distinguished cluster, but AIDS‐related DLBCL and pyothorax‐associated lymphoma formed larger clusters than other tumors. These data suggest that expression of miRNAs, including EBV‐encoded miRNAs, is associated with the tumor type and status of virus infection in these tumors.