Elucidation of the miRNome in EBV-positive and EBV-negative PTLD

Abstract

Post-transplant lymphoproliferative disorder (PTLD) is a potentially fatal complication of organ transplantation characterized by abnormal proliferation of lymphoid cells in the setting of immunosuppression after transplantation. Epstein-Barr virus (EBV) is responsible for abnormal lymphocyte proliferation in 50–80% of PTLDs, particularly in early-onset disease. The remaining PTLDs (20–50%) are EBV-negative, with etiology currently unknown. EBV was the first human virus found to encode microRNAs (miRNA). In addition to encoding miRNAs, EBV has also been found to modulate expression and regulation of host-cell-encoded miRNAs. The purpose of this study was to define the miRNome of EBV+ and EBV- PTLD. RNA was isolated from formalin-fixed paraffin-embedded tissue sections of EBV+ and EBV- PTLD tumors (n=5 for each). miRNA-microarray profiling was used to evaluate expression levels of miRNA. One-way ANOVA analysis was used to identify the miRNAs differentially modulated within the groups. 118 miRNAs in total were found to be differentially expressed, in particular 76 up-regulated and 42 down-regulated. In order to validate these initial results, RNA was isolated from additional FFPE tissue sections of EBV+ (n=10) and EBV- (n=5) PTLD tumors, and real-time qPCR was used. Thus far, three miRNAs (hsa-miR-155, hsa-miR-17, and hsa-miR-106a) have been shown to be statistically significantly regulated. Specifically, expression levels of hsa-miR-155 were 2.8-fold greater in EBV+ PTLD tumors (p=0.05). In contrast, levels of hsa-miR-17 and has-miR-106a were 2.2 fold (p=0.04) and 2.1 fold (p=0.05) higher, respectively in EBV- tumors. EBV-encoded miRNAs were also analyzed, and 35 viral miRNAs were detected in EBV+ PTLD. The EBV miRNAs include EBV-miR-BART7-3P, EBV-miR-BART1-5p, EBV-miR-BART14-3p, EBV-miR-BART8-3P, and EBV-miR-BART11-5P. In conclusion, our findings demonstrate that the miRNA profile of EBV+ PTLD tumors is distinguishable from EBV- PTLD tumors and suggest that miRNA may be useful as biomarkers in PTLD.