Epstein-Barr Virus BART9 miRNA Modulates LMP1 Levels and Affects Growth Rate of Nasal NK T Cell Lymphomas

Rajesh Ramakrishnan, Hart Donahue, David Garcia, Jie Tan, Norio Shimizu, Andrew P. Rice, Paul D. Ling

Nasal NK/T cell lymphomas (NKTCL) are a subset of aggressive Epstein-Barr virus (EBV)-associated non-Hodgkin's lymphomas. The role of EBV in pathogenesis of NKTCL is not clear. Intriguingly, EBV encodes more than 40 microRNAs (miRNA) that are differentially expressed and largely conserved in lymphocryptoviruses. While miRNAs play a critical role in the pathogenesis of cancer, especially lymphomas, the expression and function of EBV transcribed miRNAs in NKTCL are not known. To examine the role of EBV miRNAs in NKTCL, we used microarray profiling and qRT-PCR to identify and validate expression of viral miRNAs in SNK6 and SNT16 cells, which are two independently derived NKTCL cell lines that maintain the type II EBV latency program. All EBV BART miRNAs except BHRF-derived miRNAs were expressed and some of these miRNAs are expressed at higher levels than in nasopharyngeal carcinomas. Modulating the expression of BART9 with antisense RNAs consistently reduced SNK6 and SNT16 proliferation, while antisense RNAs to BARTs-7 and -17-5p affected proliferation only in SNK6 cells. Furthermore, the EBV LMP-1 oncoprotein and transcript levels were repressed when an inhibitor of BART9 miRNA was transfected into SNK6 cells, and overexpression of BART9 miRNA increased LMP-1 protein and mRNA expression. Our data indicate that BART9 is involved in NKTCL proliferation, and one of its mechanisms of action appears to be regulating LMP-1 levels. Our findings may have direct application for improving NKTCL diagnosis and for developing possible novel treatment approaches for this tumor, for which current chemotherapeutic drugs have limited effectiveness.