MicroRNA Function in Human Hematopoiesis: Identification of Lineage- and Stage-Specific Expression Profiles, Pivotal Targets and Regulatory Circuitries.

MicroRNAs (miRs) play a key role in the regulation of gene expression in normal and abnormal cell growth/differentiation, via suppression of translation or degradation of target mRNAs mainly by base pairing to their 3′ UTR sequences. However, little is known on miR expression/function in normal hematopoiesis. We have assayed the level of miRs in serum-free unilineage culture of cord blood (CB) CD34+ hematopoietic progenitor cells (HPC) through the erythroid (E), megakaryocytic (Mk), granulocytic (G) and monocytic (Mo) series at discrete stages of differentiation up to terminal maturation. The assay included control CB T cells. The analysis was performed by microarray and confirmed by Northern blot. Altogether, 49 miRs are expressed at significant levels in hematopoiesis. Cluster analysis indicates lineage-specific expression patterns. Furthermore, miR levels are mostly downmodulated through HPC differentiation/maturation. A group of 12 single or clustered miRs is sharply modulated (> 10-fold) in hematopoiesis according to specific patterns, thus suggesting their key functional role. This group includes:

• miR-223, downmodulated in the E series, but upmodulated in the other lineages;

• miR-221/222, miR-146 and miR-155 declining more sharply in E than other lineages;

• miR-17/20/106 and miR-25, miR-32, declining prevailingly in the G and Mo series;

• a miR group showing little downmodulation in the Mk or Mo lineages (miR-123, -130a or miR-181a, b, respectively);

• miR-99b, -100 downmodulated in all lineages;

• miR-210 upmodulated in all lineages.

Bioinformatic analysis suggests that these miRs have important targets, which have been in part verified by the luciferase target assay (see below). Relevant examples include miR-221/222 and -130a targeting kit receptor, miR-146 targeting SDFR/CXCR4, miR 181a, b, 99b and 100, 210 targeting diverse HOX genes (respectively, HOXB5, A1 and A3). Novel miR-based regulatory circuitries have been identified:

• (a) miR-221/222 modulate erythropoietic and erythroleukemia cell growth via kit receptor targeting (N. Felli et al, PNAS, 2005);

• (b) miR-17-5p/20a/106 function as a master gene complex controlling monocytopoiesis through AML1 targeting, leading to M-CSF receptor transactivation (L. Fontana et al, this Meeting);

• miR-155 and -221/222 control megakaryopoiesis at progenitor and precursor level through Ets-1 multitargeting (G. Marziali et al, this Meeting).