Mirna Profiling Reveals Specific Patterns for Normal B Cell Subsets and B Cell Lymphomas with a Unique Burkitt Lymphoma Profile

Abstract: MicroRNAs (miRNAs) are small (22–23nt) noncoding RNAs, which negatively regulate gene expression by inhibiting protein translation. MiRNAs play an important role in various cellular processes such as hematopoiesis. Deregulated expression is associated with development of B-cell lymphomas. The aim of this study was to define miRNA expression profiles to identify differentially expressed miRNAs in normal B cell subsets and malignancies derived from these subsets. Using Agilent Human miRNA microarrays the expression levels of 556 miRNAs were determined in 7 Mantle cell lymphoma (MCL), 7 Follicular lymphoma (FL), 7 paediatric Burkitt lymphoma (BL) and 13 Chronic lymphocytic lymphoma (CLL; 8 ZAP70 pos and 5 ZAP70 neg) cases, as well as in naïve, germinal center (GC), memory B cells and plasma cells obtained from 3 paediatric tonsils. Median normalisation was performed on all array data using GeneSpring GX 9.0.5. Differentially expressed miRNAs were defined by at least a four fold difference using ANOVA (p<0.05). Quantitive (q)RT-PCR was used for validation of the arrays. In the normal B-cell subsets 23 differentially expressed miRNAs were found with a four fold difference. Hierarchical clustering revealed a distinct pattern of these subsets. Specifically in GC B cells miR-15b, miR-17-5p and its seed family member miR- 106a were upregulated in comparison to the naïve, memory B cells and plasma cells. MiR-150, miR-221 and miR-222 were downregulated. Only miR-146a was significantly differentially expressed between naïve and memory B cell subsets. In the lymphoma cases a total of 59 miRNAs were differentially expressed. Hierarchical clustering of these miRNAs grouped all four lymphoma entities separately. ZAP70 positive and negative CLL cases did not cluster separately and none of the miRNAs were differentially expressed. Interestingly, 35 miRNAs were specifically deregulated in the Burkitt lymphomas as compared to MCL, FL and CLL, including miR-18a (up), miR-29a, miR-150, miR-155 and miR-222 (down). Almost all of these miRNAs were found not to be differentially expressed within the normal B cell subsets suggesting that these miRNAs might play a specific role in the biologic behaviour of BL. Comparison of BL to normal GC B cells revealed 15 miRNAs upregulated, including miR-126 and miR-145 (fold change >40), and 79 miRNAs downregulated, including miR-150, miR-155, miR-15, miR-16, miR- 17-5p, miR-21 and miR-222. In comparison with GC B cells FL showed 11 upregulated miRNAs, e.g. miR-143 and miR-145 (fold change >40) and 49 downregulated miRNAs. In comparison with memory B cells 21 miRNAs in the CLL group were upregulated, including miR-143 and miR-145 (fold change > 40) and 83 miRNAs were downregulated. Finally MCL was compared to the naïve B cells and showed 23 upregulated miRNAs with miR-126 showing the highest fold change (>40) and 70 downregulated miRNAs. In conclusion, we have identified specific miRNA profiles for MCL, BL, FL and CLL and also for normal B cell subsets. The differentially expressed miRNA profiles contain several miRNAs that have been shown to be directly or indirectly involved in B cell malignancies but also contain new potentially interesting miRNAs. We also have found several miRNAs that may play a tumor specific role in BL and it can be speculated that miRNAs contained in this profile may target genes related to the more aggressive clinical phenotype of BL.