Deep sequencing of miRNAs in clinical samples of Epstein-Barr virus (EBV)-associated lymphomas

Epstein-Barr virus (EBV) encodes 39 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 virus miRNA in the primary tumors of EBV-associated B-lymphoproliferative disorders. In the present study, a next generation sequencer and quantitative real-time RT-PCR were used to determine the expression profiles of miRNAs in EBV-infected cell lines and EBV-associated B-cell lymphoma, 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 sequencer revealed that EBV-encoded miRNA accounted for up to 34% of total annotated miRNA in these cases. Expression of three BHRF1 miRNAs was significantly high in AIDS-related DLBCL and pyothorax-associated lymphoma comparing with methotrexate-associated lymphoproliferative disorder and EBV-positive DLBCL of the elderly, suggesting the association of BHRF1 expression with latency III EBV infection. Heat-map/clustering analysis on the data by next generation sequencer demonstrated that AIDS-related DLBCL and EBV-positive DLBCL of the elderly formed isolated clusters from other diseases based on expression of all miRNAs including cellular and EBV miRNAs. These data suggest that expression of miRNAs including EBV-encoded miRNA is associated with the tumor type and status of virus infection in the tumor.