Methylation and Silencing of miRNA-124 In Hematopoietic Cells by EVI1

Abstract 2476

The inappropriate expression of EVI1 is associated with aggressive myelodysplastic syndromes (MDS) and acute myeloid leukemia. We previously showed that in vivo EVI1 causes an MDS-like disease in mice and that in vitro it alters cell cycling, self-renewal, and differentiation pathways of primary bone marrow (BM) cells. It is likely that these alterations contribute to the oncogenic potential of EVI1. MicroRNAs (miRNA) are small noncoding RNAs that are 21 to 25 nt in length that up- and down-regulate gene expression during cell development, proliferation, differentiation, and apoptosis. MiRNAs play a critical role in solid and hematopoietic cancers. We found that in hematopoietic cells the expression of several miRNAs is significantly altered by EVI1. Among them, miRNA-124 is strongly downregulated in EVI1-expressing primary BM and 32Dcl3 cells. To evaluate the role of miRNA-124 in primary murine BM cells, we isolated Lin- cells and infected the cells with the empty vector, EVI1, or both EVI1 and miRNA-124. We confirmed that EVI1 alters cellular pathways regulating self-renewal, S1 phase entry, and differentiation. More importantly, however, we found that when miRNA-124 is co-expressed with EVI1, the expression of proteins such as Cyclin D3 and BMI-1, that control some of these pathways, is normalized. These results suggest that the effects of EVI1 are mediated by miRNA-124 repression. We hypothesized that the down-regulation of miRNA-124 could be associated with inappropriate DNA methylation of the miRNA. Both in man and mouse, there are three known alleles of miRNA-124. In the mouse, the alleles are located on chromosome bands 2H4, 3A1, and 14D1. Analysis of the three alleles indicates that miRNA-124-3 harbors the larger number of CpG dinucleotides, including 24 CpG within a stretch of 190 bp upstream of and overlapping the stem-loop start-site. By using the bisulfite DNA sequencing method, we found that in BM cells the number of methylated CpG dinucleotides in this region is significantly increased after EVI1 expression. The DNA methylation is mostly centered in two clusters, which we named cluster I and cluster II. These data clearly show that miRNA-124 is a critical target of EVI1 in human diseases.