Modulation of IL-10-Specific B Cell MicroRNAs by Latent Membrane Protein 1 and Epstein-Barr Virus

Abstract

Post-Transplant Lymphoproliferative Disorder (PTLD) is a potentially fatal side effect of the immunosuppressive agents used to prevent allograft rejection. PTLD is associated with Epstein-Barr Virus (EBV) infection and can result in malignant B cell lymphoma. Latent membrane protein 1 (LMP1), a viral mimic of the B cell costimulatory protein CD40, is the major oncogene of EBV and can usurp host cell pathways to promote cell survival. LMP1 activates the Akt/PI3K signaling pathway to drive IL-10 production, which is essential for the autonomous proliferation of EBV+ B cell lymphomas. MicroRNAs are small, non-coding RNAs that post-transcriptionally regulate gene expression to control cellular events such as differentiation, proliferation, and cell survival. As aberrant expression of microRNAs is found in a variety of human malignancies, our goal was to determine if microRNAs participate in LMP1-induced IL-10 production in PTLD-derived cell lymphomas. To first establish that EBV infection can modulate host cell microRNA, we used Taqman microRNA qPCR arrays to detect the microRNA expression profiles of six B cell tumor lines derived from patients with PTLD, two B cell lines generated by infection with the B95.8 strain of EBV obtained from a patient with mononucleosis, and one EBV-negative B cell line. Expression data was then normalized internally and to the EBV-negative B cell line. Our analysis showed that compared to the EBV-negative B cell line, B cells infected with the benign B95.8 strain of EBV had the altered expression (2-fold) of 294 microRNAs while the PTLD-derived EBV+ B cell lines had the altered expression of 224 microRNAs. Twenty-eight microRNAs were altered in both cases of EBV infection. To examine the specific role of LMP1 in host cell microRNA expression, we used an inducible system of LMP1 activation and expressed different LMP1 molecules in an EBV negative B cell lymphoma line. Activation of the B95.8 form of LMP1 modulated 62 cellular microRNAs and 142 microRNAs were modulated by all five of the PTLD-derived LMP1 molecules. Notably, only 28 microRNAs were modulated by both benign and tumor LMP1. The microRNAs aberrantly regulated by either EBV infection or LMP1 activation were next analyzed with target prediction software for potential binding to the 3″UTR of IL-10. MicroRNA-21, -106A, -106B, -194, -340, -374 and -374-5p fit these criteria and were tested for direct binding to the 3″UTR of IL-10 in luciferase reporter assays. The overexpression of microRNA-106A, -106B, -194, -374, -374-5p each negatively regulated the IL-10 3″UTR. Conversely, microRNA-21 overexpression positively modulated the 3″UTR of IL-10 while microRNA-340 overexpression had no effect. Together, these findings indicate that EBV infection and LMP1 activation induce host cell microRNAs that regulate production of the autocrine growth factor IL-10 in EBV+ B cell lymphomas. Thus, cellular microRNAs are a potential target for inhibiting the growth of PTLD-associated B cell lymphomas.