Global miRNA expression status of human hematopoietic stem–progenitor cells modulates myelopoiesis

We investigated how suppression of the most upstream microRNA–processing RNase, Drosha, affects the differentiation of human CD34+ hematopoietic stem–progenitor cells (HSPCs). We hypothesized that knock-down of Drosha would alter blood lineage development by modulating the expression of microRNAs. Lentiviral delivery to HSPCs of a short-hairpin targeting Drosha resulted in a viable phenotype with promotion of myeloid, and especially monocytic, maturation and suppression of apoptosis. Our results show that Drosha deficiency triggered a parallel upregulation of components of the RNAi machinery, including DGCR8, Dicer and Ago2. Deep sequencing analyses revealed global miRNA deficiency after Drosha short-hairpin treatment with relative maintenance of mature miR-223 expression. Restoration of miR-223 to normal levels after Drosha knock-down further enhanced monocytic maturation concomitant with the modulation of myeloid transcription factors that promoted monocytic differentiation. Our results support a miRNA accentuation model in which relative enhancement of miR-223 increases levels of PU.1 thereby promoting monocytic differentiation. Overall design: CD34+ HSPCs were isolated from umbilical cord blood and transduced with an empty lentivector (EV) or a lentivector encoding a short-hairpin RNA targeting the pri-miRNA–processing enzyme, Drosha (shDrosha). EV and shDrosha transduced HSPCs were grown in liquid culture promoting myelopoiesis and sampled on days 0 and 7 for total RNA collection. Total RNA was size fractionated to enrich for the small RNA population and deep sequenced using ABI''s SOLiD 4.0 platform.