BCR-ABL negative myeloproliferative neoplasms (MPNs; polycythemia vera, essential thrombocythemia, primary myelofibrosis) are malignant diseases arising from a multipotent hematopoietic progenitor, frequently altered by JAK2 V617F or other JAK/STAT activating mutations. The thrombopoietin receptor (TpoR, MPL) is one of the major dimeric cytokine receptors that use JAK2 in the myeloid lineage, and was found to be down-modulated in certain MPN patients. We searched for negative regulators of MPL expression. Here we report that miR-28 targets the 3 untranslated (3UTR) region of MPL, inhibiting its translation, as well as other proteins potentially involved in megakaryocyte differentiation, such as E2F6. Expression of miR-28 in CD34-derived megakaryocytes inhibited terminal differentiation. miR-28 was found to be overexpressed in platelets of a fraction of MPN patients, while it was expressed at constant low levels in platelets from healthy subjects. Constitutive activation of STAT5 leading to autonomous growth of hematopoietic cell lines was associated with increased miR-28 expression. We discuss how down-modulating MPL and other targets of miR-28, and of related miR-708 and miR-151, could contribute to MPN pathogenicity. (Blood. 2010;116(3): 437-445) IntroductionThe BCR-ABL negative myeloproliferative neoplasms (MPNs), polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) are malignant diseases arising from a mutant multipotent hematopoietic stem cell (HSC), 1,2 that are associated with constitutively active JAK-STAT signaling. The JAK2 V617F mutation is present in 95% of PV and 50% of ET and PMF. [3][4][5][6] JAK2 exon 12 mutations are found in a minority of PV patients. 7 5% of PMF and 1% of ET harbor thrombopoietin receptor (TpoR, MPL) W515 mutations, 8,9 which constitutively activate JAK2 signaling. 10 Moliterno et al have reported diminished platelet MPL expression in MPNs 11 and an inverse correlation between JAK2 V617F allele burden and MPL expression, although down-modulation of MPL was observed also in JAK2 V617F-negative patients. 12 This suggests that mechanisms that limit the expression, surface localization, and function of MPL might operate during the establishment of MPNs.We searched for putative microRNAs 13 that target the 3ЈUTR of the MPL mRNA. We found that miR-28 is an inhibitor of MPL translation, which is also the case for 2 close relatives of miR-28, miR-151 and miR-708. We identified several miR-28 targets, besides MPL, such as E2F6, a transcription factor involved in the control of proliferation and apoptosis, and the MAP-kinase MAPK1/ ERK2. We then detected induction of miR-28 in cell lines transformed by JAK2 V617F or activated MPL mutants. We have also investigated levels of expression of miR-28 in platelets from healthy subjects and MPN patients. Methods Cell lines, plasmid/luciferase constructs, and reagentsHuman erythroleukemia (HEL), JAK2-deficient cell ␥2A, UT-7, Mo-7e, and Ba/F3 cells were maintained as described. [14][15][16][17][18] The UKE-1 ce...

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