Rontauroli Sebastiano, Norfo Ruggiero, Pennucci Valentina, Zini Roberta, Ruberti Samantha, Bianchi Elisa, Salati Simona, Prudente Zelia, Rossi Chiara, Rosti Vittorio, Guglielmelli Paola, Barosi Giovanni, Vannucchi Alessandro, Tagliafico Enrico, Manfredini Rossella
Centre for Regenerative Medicine, Life Sciences Department, University of Modena and Reggio Emilia, Modena, Italy.
Center for The Study of Myelofibrosis, Biotechnology Research Area, IRCCS Policlinico S. Matteo Foundation, Pavia, Italy.
Oncotarget. 2017 Mar 28;8(13):21380-21397. doi: 10.18632/oncotarget.15226.
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.
原发性骨髓纤维化（PMF）是一种慢性费城染色体阴性骨髓增殖性肿瘤，其特征为造血干细胞来源的克隆性骨髓增殖，尤其累及巨核细胞系。为了更好地描述微小RNA表达改变如何促进PMF发病机制，我们之前对PMF造血干/祖细胞（HSPC）的基因和微小RNA表达谱进行了综合分析，这使我们能够鉴定出miR-494-3p为上调的微小RNA，预计其靶向的下调mRNA数量最多。为了阐明miR-494-3p在造血分化中的作用，在本研究中我们证明，在正常HSPC中强制表达miR-494-3p可促进巨核细胞生成。miR-494-3p过表达后的基因表达谱分析能够鉴定出在微小RNA过表达后以及PMF CD34+细胞中均普遍下调的基因。其中，细胞因子信号传导抑制因子6（SOCS6）通过荧光素酶测定被确认为miR-494-3p的靶标。蛋白质印迹分析显示，在过表达miR-494-3p的细胞中，SOCS6蛋白水平降低以及STAT3激活。此外，在HSPC中瞬时抑制SOCS6表达表明，SOCS6沉默可刺激巨核细胞生成，模拟了miR-494-3p过表达时观察到的表型效应。最后，为了揭示miR-494-3p上调对PMF发病机制的作用，我们在PMF HSPC中进行了抑制实验，结果显示miR-494-3p沉默导致SOCS6上调并损害巨核细胞分化。综上所述，我们的结果首次描述了miR-494-3p在正常HSPC分化过程中的作用，并表明其表达增加以及随后其靶标SOCS6的下调可能导致PMF患者中常见的巨核细胞增生。
