“MiRNA expression in Primary Myelofibrosis: characterization and pathophysiology implications”

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by megakaryocyte (MK) hyperplasia, bone marrow fibrosis, and abnormal stem cell trafficking. PMF may be associated with somatic mutations, about 90% of patients harbor one of three “driver” mutations, with mutational frequencies of approximately 60%, 22% and 6% for JAK2, CALR and MPL, respectively. Other “non-driver” mutations have also been described in PMF involving different cellular targets such as epigenetic regulatory pathways genes (ASXL1, DNMT3A, EZH2, IDH1 and IDH2, TET2), splicing factor genes (SRSF2, SF3B1) and progression to leukemia (CBL, LNK, RUNX1, TP53). However, several aspects of its pathogenesis remain elusive. Increasing evidences indicate that the deregulation of microRNAs (miRNAs) might plays an important role in hematologic malignancies, including MPN. In this work, in collaboration with a group at the Hemopoietic Stem Cells Laboratory of the University of Modena and Reggio Emilia and a bioinformatics group of University of Padua, it was performed a genome wide analysis of coding RNA (GEP) and microRNA (miEP) expression in CD34+ cells purified from patients with PMF and from health subjects initially using Affymetrix technology. By means of miRNA-gene expression integrative analysis, it was found different regulatory networks involved in the dysregulation of transcriptional control and chromatin remodeling. In particular, it was identified a network gathering several miRNAs with oncogenic potential (e.g. miR-155-5p) and targeted genes whose abnormal function has been previously associated with myeloid neoplasms, including JARID2, NR4A3, CDC42, and HMGB3. Because the validation of miRNA-target interactions unveiled JARID2/miR-155-5p as the strongest relationship in the network, it was studied the function of this axis in normal and PMF CD34+ cells. This study showed that JARID2 downregulation mediated by miR-155-5p overexpression leads to increased in vitro formation of CD41+ MK precursors. These findings suggest that overexpression of miR-155-5p and the resulting downregulation of JARID2 may contribute to MK hyperplasia in PMF. At the same time, to attain deeper and more extensive knowledge of short RNAs (sRNAs) expression pattern in CD34+ cells and of their possible role in mediating post-transcriptional regulation in PMF, CD34+ cells from healthy subjects and PMF patients were sequenced with Illumina HiSeq2000 technology. It was detected the expression of 784 known miRNAs, with a prevalence of miRNA up-regulation in PMF samples, and discovered 34 new miRNAs and 99 new miRNA-offset RNAs (moRNAs) in CD34+ cells. Thirty-seven small RNAs were differentially expressed in PMF patients compared with healthy subjects, according to microRNA sequencing data. Five miRNAs (miR-10b-5p, miR-19b-3p, miR-29a-3p, miR-379-5p, and miR-543) were deregulated also in PMF granulocytes. Moreover, 3’-moR-128-2 resulted consistently downregulated in PMF according to RNA-seq and qRT-PCR data both in CD34+ cells and granulocytes. Target predictions of these validated small RNAs de-regulated in PMF and functional enrichment analyses highlighted many interesting pathways involved in tumor development and progression, such as signaling by FGFR and DAP12 and Oncogene Induced Senescence. As a whole, data obtained in this study deepened the knowledge of miRNAs and moRNAs altered expression in PMF CD34+ cells and allowed to identify and validate a specific small RNA profile that distinguishes PMF granulocytes from those of normal subjects. It was thus provided new information regarding the possible role of miRNAs and, specifically, of new moRNAs in this disease. The expression of 175 miRnas in plasma samples was also analyzed in the patients with PMF and the healthy donors and it was identified the presence of 6 differentially expressed miRNAs deregulated in significant statistically way (P value <0.05): miR-let7b*, miR-10b-5p, miR-424 and miR-99a were resulted up-regulated instead miR-144* and miR-375 were down-regulated in PMF patients. These data show a distinct plasma miRNA expression patterns in patients with PMF compared with health subjects which could have a potential utility as prognostic biomarkers. Finally, in order to clarify the contribution of microRNAs also in to the pathogenesis of JAK2V617F-positive MPNs, it was analysed the miRNAs expression pattern in erythroid (TER119+) and mieloid (GR1+) cells purified from BM of JAK2V617F knock-in (KI) mouse model using TaqMan® Real time PCR. In this part of the study, it was identified a list of differentially expressed miRNAs also in JAK2V617F KI mouse whose deregulation might contribute to the development and phenotype of MPNs. The results of this work provided novel data regarding the expression profile of small RNA expressed in CD34+, granulocytes and plasma of PMF patients; in addition, for the first time a new moRNAs was described as possible contributors to disease pathogenesis. Finally, it was identified a list of differentially expressed miRNAs in JAK2V617F KI mouse whose deregulation might contribute to the development/phenotype of MPNs. This information may represent the basis for further studies aimed at a deeper knowledge of the prognostic and therapeutical role of miRNAs and also moRNAs in PMF.