Integrated analysis of microRNA and mRNA expression profiles in physiological myelopoieis: role of hsa-miR-299-5p in CD34+ progenitor cells commitment

Cell fate decisions in the hematopoietic system appear to be directed by an antagonistic or synergistic interplay of transcription factors that pivot immature blood progenitors for cell specification. Multipotent progenitors initially trigger a promiscuous transcriptional program and, as soon as they commit to a restricted fate, they reinforce unilineage gene expression and withdraw transcripts affiliated with alternative blood cell types. MicroRNAs appear to be especially pertinent in driving this particular behavior representing a new component of the hematopoietic gene regulatory network. In fact, the archetypal microRNA can potentially regulate hundreds of genes even if most targets contain isolated microRNA recognition sites that may be inadequate for complete gene silencing. According to Bartel’s theory, microRNAs mediated post-transcriptional control offers a more flexible and rapid way of tuning genes compared to transcriptional control (Bartel DP and Chen CZ, Nat Rev Genet 2004). These issues encouraged some investigators to explore the association of microRNAs and genes expression profiles obtained from the same cell type and advocated that microRNAs evolved to regulate gene expression programs and remove gene products unnecessary or potentially dangerous more rapidly than might occur by natural decay. Although many studies addressed the role of microRNAs during the normal myeloid differentiation process, only Georgantas and co-workers focused onto the impact of microRNAs on mRNA expression levels but limited the analyses to data obtained from human CD34+ stem/progenitor cells (Georgantas RW 3rd et al, PNAS 2007). In order to shed light onto the interplay of mRNAs and microRNAs during the normal myeloid commitment and verify that increased expression of a microRNA is skillful to modulate the levels of corresponding target mRNAs, we obtained microRNAs profiles from CD34+ hematopoietic progenitor cells (CD34 HPCs) and in-vitro differentiated precursors: erythroblasts, megakaryoblasts, monoblasts and myeloblasts (ERY, MKC, MONO and MYELO). We therefore analyzed these microRNA expression profiles together with the gene expression profiles of the same populations and observed that for the most part of the microRNAs specifically up-regulated in one single progeny an inverse correlation between microRNAs and down-regulated putative targets expression levels occurs, i.e. down-regulated genes showed an enrichment for the conserved putative targets of up-regulated microRNA. Among these microRNAs, hsa-miR-299-5p emerged as an interesting candidate to demonstrate how the integrated analysis of microRNA and mRNA expression data can help shedding light on the regulatory mechanisms governing cell differentiation. In particular, we used hsa-miR-299-5p to prove that the forced expression of a single lineage-specific microRNA is able to control the cell fate of CD34 HPCs grown in multilineage culture conditions. Clonogenic and liquid culture differentiation assays after gain- and loss-of-function experiments revealed that indeed hsa-miR-299-5p regulates hematopoietic progenitors fate modulating megakaryocytic-granulocytic versus erythroid-monocytic development.