MiRNA profiling in B non‐Hodgkin lymphoma: a <i>MYC</i>‐related miRNA profile characterizes Burkitt lymphoma (2010)

Several studies have indicated the importance of miRNAs in B cell maturation and in the development of B cell lymphomas. The oncogene MYC plays an important role in B cell lymphomagenesis, particularly in Burkitt lymphoma (BL). Several recent publications have shown that MYC regulates expression of up to 60 miRNAs (Tables I and SI). The impact of the translocation and overexpression of MYC on the miRNA profile in BL has not yet been explored. We determined the miRNA expression profile of paediatric t(8;14) positive and high MYC expressing BL in comparison to MYC translocation negative mantle cell lymphoma (MCL), follicular lymphoma (FL) and chronic lymphocytic leukaemia (CLL). As a control we included normal B cell subsets obtained from hyperplastic tonsils. Hierarchical clustering showed that the B cell subsets and the non-Hodgkin lymphomas (NHLs) formed two distinct sub-clusters (Fig 1). Unsupervised clustering of the 23 miRNAs significantly differentially expressed between the four B cell subsets (>4-fold) revealed one cluster for the naïve and memory B cells and two additional clusters for the germinal centre (GC) B cells and plasma cells. These results were consistent with previously published data (Appendix S1). 76 miRNAs were differentially expressed (>4-fold) between the four NHL subtypes. Most differences were observed between BL and the other NHLs (CLL n = 58, FL n = 32 and MCL n = 36 miRNAs). Unsupervised hierarchical clustering analysis revealed a unique miRNA profile in BL (Appendix S2). In contrast, a maximum of eight miRNAs were differently expressed between MCL, FL and CLL. A list of validated target genes of the differentially expressed miRNAs is presented in Table SII. Comparing each malignancy to its normal counterpart (MCL with naïve, FL and BL with GC B cells and CLL with memory cells), 54–77 miRNAs were differentially expressed (Figure S1). Heatmap of the unsupervised hierarchical clustering of all miRNAs and miRNAs that are MYC-regulated. MiRNA profiles were generated using Agilent Oligo Microarray Kit (G4470A; Agilent, Santa Clara, CA, USA) and the protocol provided by the manufacturer. In total, 256 out of the 556 human miRNAs were expressed in at least three out of the 44 samples. (A) Heatmap of 256 miRNAs expressed in at least three samples. (B) Enlargement of the clustering tree. With the exception of one FL sample (#FL2) all normal B cell samples cluster separately from the lymphoma samples. (C) Box plot of the MYC expression levels in BL, CLL MCL determined by qRT-PCR. High MYC expression levels were observed in all six MYC translocation-positive BL, whereas the MYC levels were much lower in MCL, CLL and FL.). (D) Heatmap of the MYC induced miRNAs; MYC induced and repressed miRNAs are indicated on the right-hand side. Of the 60 reported MYC regulated miRNAs (Table I), 50 were expressed in at least three of the NHL samples and therefore considered to be evaluable. A significant difference between the high MYC BL and the other low MYC lymphoma samples was observed for 39 miRNAs. The miRNA pattern in BL is consistent with the expected MYC regulation. CLL showed an almost inversed pattern. MCL and FL showed intermediate pattern. *, indicates miRNAs with a proven MYC binding site in the promoter region. MYC expression has been shown to be a dominant factor in the regulation of many miRNAs. In the present series of lymphomas, quantitative reverse transcription polymerase chain reaction (qRT-PCR) showed a much higher expression in BL than all other NHL (Fig 1C). Comparison of the MYC-high BL to all other MYC-low lymphomas revealed 122 differentially expressed miRNAs (Table SIII), including 39 of the 50 evaluable (78%) known targets of MYC (10 miRNAs were not expressed in our cases) (Fig 1D and Table I). The expression level of these 39 miRNAs was always consistent with the expected up- or downregulation, with most miRNAs being downregulated. This indicates a dominant MYC-induced miRNA profile in primary BL. This signature included 39 of the known MYC-regulated miRNAs. The 83 other miRNAs were also differentially expressed between MYC-high BL and MYC-low NHL samples. At present, there are no data supporting a direct MYC-dependent regulation for those 83 miRNAs. A MYC-dependent miRNA signature has also been suggested for diffuse large B cell lymphoma based on a differential expression of MYC targets (Li et al, 2009). Four BL cases (B4, B5, B6 and B7) and two MCL cases (M2 and M3) harboured a 13q31.3 amplification (determined by fluorescence in situ hybridization, results not shown). These six cases showed high expression of the miR-17-92 cluster (Fig 1D). Both in BL and MCL, this association was independent of the MYC expression level (Fig 1C). In line with the findings of Tagawa et al (2007) this indicates that genomic amplification and not MYC overexpression is instrumental in the expression of the miR-17-92 cluster. The miR-17-92 cluster is positively regulated by MYC and acts with MYC to accelerate tumour development (He et al, 2005). MiR-17 and miR-20, two members of this cluster, promote cell cycle progression via E2F1 (O’Donnell et al, 2005). One interesting MYC-repressed gene is MIRLET7A. As MYC is a direct target of MIRLET7A this suggests a positive stimulatory loop for MYC (Sampson et al, 2007). Induction of MIRLET7A in the Namalwa BL cell line resulted in reduced MYC expression and reduced proliferation, whereas downregulation of MYC resulted in increased expression of MIRLET7A. In addition to a significant downregulation of MIRLET7A, we also observed a significant downregulation for MIRLET7E in BL compared to the three other NHL subtypes. MiR-150 was also significantly downregulated in BL and targets MYB, which has an essential role in haematopoietic and lymphoid development and apoptosis (Xiao et al, 2007). Overexpression of miR-150 in BL cells resulted in reduced MYB levels and increased apoptosis (Tan et al, 2009). Interestingly miR-15a has recently also been shown to repress the MYB oncogene in leukemic cell lines (Zhao et al, 2009). Low expression of miR-150 in BL compared to the other three NHL subtypes and of miR-15a in comparison to CLL might thus result in enhanced MYB levels in BL. Ectopic expression of miR-26a in BL cell lines impaired cell cycle progression via its target EZH2, a member of the polycomb-group of genes (Sander et al, 2008). The low expression of miR-26a in BL is thus consistent with the previously observed high expression of EZH2 in BL. Concurrent with previous reports, we found downregulation of miR-155 in BL. Although it is unclear which target gene(s) promote tumorigenesis in lymphomas, loss of the miR-155 dependent regulatory control of activation-induced cytidine deaminase (AID) expression, by the introduction of a point mutation in the miR-155 binding site, resulted in increased levels of AID and a higher frequency of MYC translocations (Dorsett et al, 2008). This is consistent with low miR-155 levels in MYC/8q24 translocation-positive BL. Finally, several other MYC-regulated miRNAs have been implicated in B cell lymphoma and were found to be downregulated in BL e.g. miR-23a/b targeting glutaminase, miR-125b targeting IRF4 and PRDM1/BLIMP1, miR-146a targeting IRAK1 and TRAF6 and miR-223 targeting LMO2 (Table SII). To conclude, this study showed a very characteristic MYC-induced miRNA expression profile in BL. This indicates that MYC regulates BL cell fate in a direct mode at the transcriptional level and indirectly at the translational level by influencing the miRNA profile. Our data support previous studies showing MYC-induced miRNA changes in cell lines and animal models. Fig S1. Heatmap of the miRNAs differentially expressed (>4-fold) between the lymphomas and their postulated normal counterparts. Table SI. Overview of known c-Myc regulated miRNAs and differential expression between BL versus CLL, MCL and FL. Table SII. Overview of validated target genes for differentially expressed miRNAs. Table SIII. Overview of differentially expressed miRNAs between Myc high BL and all other Myc low lymphomas. Appendix S1. B cell subsets. Appendix S2. NHL subsets. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.