MiR-494-3p overexpression promotes megakaryocytopoiesis in primary myelofibrosis hematopoietic stem/progenitor cells by targeting SOCS6.

Sebastiano Rontauroli,Enrico Tagliafico,Samantha Ruberti,Simona Salati,Rossella Manfredini,Vittorio Rosti,Zelia Prudente,Ruggiero Norfo,Chiara Rossi,Alessandro Vannucchi,Paola Guglielmelli,Elisa Bianchi,Roberta Zini,Valentina Pennucci,Giovanni Barosi

doi:10.18632/oncotarget.15226

Abstract

Primary myelofibrosis (PMF) is a chronic Philadelphia-negative myeloproliferative neoplasm characterized by hematopoietic stem cell-derived clonal myeloproliferation, involving especially the megakaryocyte lineage. To better characterize how the altered expression of microRNAs might contribute to PMF pathogenesis, we have previously performed the integrative analysis of gene and microRNA expression profiles of PMF hematopoietic stem/progenitor cells (HSPCs), which allowed us to identify miR-494-3p as the upregulated microRNA predicted to target the highest number of downregulated mRNAs.To elucidate the role of miR-494-3p in hematopoietic differentiation, in the present study we demonstrated that miR-494-3p enforced expression in normal HSPCs promotes megakaryocytopoiesis. Gene expression profiling upon miR-494-3p overexpression allowed the identification of genes commonly downregulated both after microRNA overexpression and in PMF CD34+ cells. Among them, suppressor of cytokine signaling 6 (SOCS6) was confirmed to be a miR-494-3p target by luciferase assay. Western blot analysis showed reduced level of SOCS6 protein as well as STAT3 activation in miR-494-3p overexpressing cells. Furthermore, transient inhibition of SOCS6 expression in HSPCs demonstrated that SOCS6 silencing stimulates megakaryocytopoiesis, mimicking the phenotypic effects observed upon miR-494-3p overexpression. Finally, to disclose the contribution of miR-494-3p upregulation to PMF pathogenesis, we performed inhibition experiments in PMF HSPCs, which showed that miR-494-3p silencing led to SOCS6 upregulation and impaired megakaryocyte differentiation.Taken together, our results describe for the first time the role of miR-494-3p during normal HSPC differentiation and suggest that its increased expression, and the subsequent downregulation of its target SOCS6, might contribute to the megakaryocyte hyperplasia commonly observed in PMF patients.

Highlights

Philadelphia-negative chronic myeloproliferative neoplasms (MPNs) are a heterogeneous spectrum of clonal hematological malignancies that include polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis, which can be primary (PMF) or secondary to PV or ET [1]
Our results describe for the first time the role of miR-4943p during normal hematopoietic stem/progenitor cells (HSPCs) differentiation and suggest that its increased expression, and the subsequent downregulation of its target suppressor of cytokine signaling 6 (SOCS6), might contribute to the megakaryocyte hyperplasia commonly observed in Primary myelofibrosis (PMF) patients
We describe for the first time the biological function of miR-494-3p in normal HSPC differentiation, demonstrating that its enforced expression leads to increased production of megakaryocytic cells and providing evidence that its phenotypic effects are mediated by SOCS6 downregulation

Summary

Introduction

Philadelphia-negative chronic myeloproliferative neoplasms (MPNs) are a heterogeneous spectrum of clonal hematological malignancies that include polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis, which can be primary (PMF) or secondary to PV or ET [1]. MPNs arise from the mutation of a single hematopoietic stem cell (HSC), which following clonal expansion leads to the overt clinical phenotype [2, 3]. While PV is mainly characterized by an overproduction of erythrocytes, ET and PMF patients show increased numbers of dysplastic megakaryocytes in the bone marrow (BM) [4, 5]. Despite the clinical heterogeneity of MPN patients, PV, ET and PMF share common genomic lesions. Several other identified lesions (i.e. loss-of-function mutations in EZH2, ASXL1, TET2, JARID2) are less frequent and usually co-occur alongside the afore-mentioned hits [11]

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Conclusion