Lineage-Specific Expression and Functional Relevance of MicroRNA Genes in Normal Hematopoiesis.

Abstract

MicroRNAs (miRs) are a class of a small (~ 22nt) RNAs, which play an important role in the negative regulation of gene expression by base-pairing to complementary sites on the target mRNAs. While it is established that miRs are involved in a variety of basic processes, e.g., cell proliferation and apoptosis, neural development, fat metabolism and stress response, little is known on their expression and function in hematopoiesis. In order to investigate miR expression in erythropoietic (E), megakaryocytic (Mk), granulopoietic (G) and monocytopoietic (Mo) lineages, we have assayed their level at discrete sequential stages of the E, Mk, G or Mo series in unilineage differentiation/maturation cultures of cord blood (CB) CD34+ cells. The analysis was performed using a microarray chip containing as probes gene-specific 40mer oligonucleotides, generated from 161 human and 84 mouse precursors miRs (Liu GC et al., PNAS, 2004). Northern blot analysis confirmed the microarray data. The results indicate that the majority of the analyzed miRs is not expressed in CB hematopoietic cells. However, 49 miRs are expressed at significant levels in CD34+ cells: in most cases the expression level declines during hematopoietic differentiation according to diverse patterns, i.e., the decline may be more or less pronounced, more or less rapid and differ in the diverse hematopoietic lineages. As expression pattern examples, we observed that: (a) miR 223 is strongly downmodulated in the E lineage, whereas its level is not affected or increased in the other series; (b) miR 221 and 222 level sharply declines in the E lineage, while the drop is less pronounced in the Mk, G and Mo series; conversely, (c) miR 17, 20, 106 are downmodulated prevalentely in the G/Mo series, as compared to the E/Mk lineages. Interestingly, cluster analysis indicates that miR expression in hematopoietic cells is sharply different from that observed in CB T lymphocytes. The lineage- and stage-specific pattern of miR expression is of functional relevance. As an example, transfection of miR 222 oligonucleotide into CD34+ cells grown in multilineage clonogenic culture causes a pronounced shift from E to GM colony formation, indicating modulation of the lineage commitment of hematopoietic progenitors. The target genes of miRs expressed in hematopoietic cells are often of pivotal functional significance, e.g., miR 222 targets the kit receptor (N. Felli et al., this Meeting). A single miR may target diverse mRNAs, e.g., miR 222 targets kit, Ets1 and Fli1. Conversely, a single mRNA may be targeted by different miRs, e.g., kit is targeted by miR 146, 221 and 222. Noterworthily, the miR expression pattern in primitive hematopoietic cells and their progeny is fully distinct from that observed in primitive mesenchymal and neural cells (i.e., “neurospheres”) and their progeny: this suggests that miR downmodulation during differentiation of primitive cells contributes to tissue-specific gene expression by unblocking translational repression of the target mRNAs.