C-Myb Restrains Megakaryopoiesis through the Hsa-MiR-486-3p-Driven Down-Regulation of C-Maf

INTRODUCTION The transcription factor c-Myb plays a key role in human primary CD34+ hematopoietic progenitor cells (HPCs) lineage choice, by enhancing erythropoiesis at the expense of megakaryopoiesis. We previously demonstrated that c-Myb affects erythroid versus megakaryocyte lineage decision in part by transactivating KLF1 and LMO2 expression. To further unravel the molecular mechanisms through which c-myb affects lineage fate decision, we profiled the miRNA and mRNA changes in myb-silenced CD34+ HPCs. METHODS RNA from CD34+ HPCs transfected with c-myb-targeting/non targeting control synthetic siRNAs was collected 24 hours post-Nucleofection for a set of 5 independent experiments. mRNA and miRNA expression for each sample were profiled by Affymetrix U219 GeneAtlas and Exiqon Human miRNome PCR Panel, respectively. miRNA/mRNA data were integrated by Ingenuity Pathway Analysis. The effects of hsa-miR-486-3p overexpression and c-Maf silencing on CD34+ cells differentiation ability were studied by morphological and immunophenotypic analyses after liquid culture and by collagen-based clonogenic assay. Furthermore, gene expression changes in CD34+ cells upon hsa-miR-486-3p overexpression were profiled by Affymetrix U219. RESULTS The integrative analysis of miRNA/mRNA expression changes upon c-myb silencing in human CD34+ HPCs highlighted a set of 19 miRNA with 150 anticorrelated putative target mRNAs. Among the miRNAs down-regulated in myb-silenced progenitors with the highest number of predicted target mRNAs, we selected hsa-miR-486-3p based on the in vitro effects of its overexpression on HPCs commitment. Indeed, morphological and flow cytometric analyses after liquid culture showed that hsa-miR-486-3p overexpression in HPCs enhanced erythroid and granulocyte differentiation while restraining megakaryocyte and macrophage differentiation. Moreover, collagen-based clonogenic assay demonstrated a strong impairement megakaryocyte commitment upon hsa-miR-486-3p-overexpression in CD34+ cells. Gene expression profiling of hsa-miR-486-3p overexpressing CD34+ cells enabled us to identify a set of 8 genes down-regulated and computationally predicted, putative hsa-miR-486-3p targets. Among them, we selected c-maf transcript as up-regulated upon myb silencing. Worth of note, c-maf silencing in CD34+ progenitor cells was able to reverse the affects of myb silencing on erythroid versus megakaryocyte lineage choice. CONCLUSIONS Integrative miRNA/mRNA analysis highlighted a set of miRNAs and anticorrelated putative target mRNAs modulated upon myb silencing, therefore potential players in myb-driven HPCs lineage choice. Among them, we demonstrated the hsa-miR-486-3p/c-maf pair as partially contributing to the effects of myb on HPCs commitment. Therefore, our data collectively identified myb-driven hsa-miR-486-3p up-regulation and subsequent c-maf down-regulation as a new molecular mechanism through which c-Myb favours erythropoiesis while restraining megakaryopoiesis.