Gene expression profile (GEP) of miR-34a-5p-overexpressing CD34+ HPCs

Primary Myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by hyperplastic megakaryopoiesis and myelofibrosis. Through a gene expression profile (GEP) study we recently highlighted the upregulationof miR-34a-5p in PMF versus healthy donor (HD) CD34+ hematopoietic progenitor cells (HPCs). To shed some light into the role of miR-34a-5p in PMF pathogenesis, here we unravelled the effects of the overexpression of miR-34a-5p in HPCs forcing its expression in HPCs. We showed that enforced expression of miR-34a-5p blocks proliferation and favours the megakaryocyte and monocyte/macrophage commitment of HPCs. Interestingly, we identified lymphoid enhancer-binding factor 1 (LEF1) and nuclear receptor subfamily 4, group A, member 2 (NR4A2) transcripts as miR-34a-5p-targets downregulated after miR-34a-5p overexpression in HPCs as well as in PMF compared with HD HPCs. Remarkably, the knockdown of NR4A2 in HPCs mimicked the antiproliferative effects of miR-34a-5p overexpression, while the silencing of LEF1 phenocopied the effects of miR-34a-5p overexpression in HPCs lineage choice, by stimulating the megakaryocyte and monocyte/macrophage commitment. Human CD34+ cells were transfected by using the 4D-Nucleofector™ System (Lonza). Briefly, starting from the day after CD34+ cells purification, each sample was electroporated twice, once every 24 hours, with 3 µg of mirVana™ miR-34a-5p mimic or mirVana™ miRNA mimic Negative Control (NegCTR mimic). For each electroporation, 4×105 CD34+ cells were resuspended in 100 µL of P3 Primary Cell Solution (Lonza) containing 3 µg of miR-34a-5p mimic or NegCTR mimic and pulsed with the program DS112. Gene expression profile (GEP) was performed on total RNA derived from three independent experiments at 24h after the last nucleofection.