Differential Expression of miRNAs in Polycythemia Vera Peripheral Blood Cells.

Polycythemia vera (PV) is a clonal myeloproliferative disorder caused by somatic mutation of a hematopoietic multipotent cell. PV hematopoiesis is characterized by an accumulation of phenotypically normal erythrocytes with overproduction of leukocytes and platelets. A somatic JAK2 V617F point mutation occurs in the majority (>95%) of PV patients. However, this mutation is also found in ∼50% of patients with either essential thrombocythemia (ET) or idiopathic myelofibrosis (MF). The non-specificity of this mutation, the presence of JAK2 V617F-negative PV patients, JAK2 V617F-negative and positive relatives, and evidence of both JAK2 V617F-negative and positive PV clones demonstrate that the JAK2 V617F mutation is not the initial and sole somatic event for the pathogenesis of PV. MicroRNAs (miRNAs) have been shown to be important regulators of hematopoiesis. To identify deregulated miRNAs involved in PV pathogenesis, we studied gene expression of miRNAs of in vitro expanded erythroid progenitors (EPs), peripheral blood mononuclear cells (MNCs), granulocytes, reticulocytes, and platelets from PV patients and healthy controls. Initially, we performed gene expression profiling in 5 PV patients and 5 control cells using CombiMatrix MicroRNA CustomArray with 326 probes. The array data were analyzed by Genesis software to determine differentially expressed miRNAs in PV. These miRNAs were tested in a larger set of samples (n=18) by qRT-PCR and their expression was correlated to the JAK2 V617F mutational level. Hierarchical clustering analysis defined miRNA expression profiles of particular cell lineage for normal and PV cells. Further, ANOVA identified 31 miRNAs differently (P<0.05) expressed in at least some PV lineage cells. Of these miRNAs, we confirmed downregulation of miR-150 in expanded EPs of all stages of maturation, downregulation of let7a and upregulation of miR-182 in PV granulocytes; upregulation of miR-143, miR-145 and miR-223 in PV MNCs; and down-regulation of miR-30b, miR-30c and miR-150 in PV reticulocytes by qRT-PCR. Correlation analysis of miRNA expression with JAK2 V617F mutational level showed a positive correlation of miR-143 (r=0.68) and a negative correlation of let7a (r =−0.63), miR-30c (r=−0.74), miR-342 (r=−0.66) and miR-150 (r=−0.89). To validate PV specificity, we compared expression levels of these miRNAs to other MPD disorders (MF and ET) in which the JAK2 V617F mutation occurs. Putative miRNA targets were predicted by TargetScan 4.0 and PicTar software, and transcript levels of selected target genes are being analyzed to determine their potential deregulation at the mRNA level. Downregulated miR-150 is predicted to the target MYB oncogene that plays an important role in erythropoiesis by maintaining proliferation at the early stages. The most potential target of let7a is HGMA2, whose aberrant expression may contribute to clonal hematopoiesis in PNH. The verification of these predictions is in process by use of miRNA inhibitors, protein levels, and functional studies of the progenitors. Our study demonstrates that the specific signatures of miRNAs define particular peripheral blood cell lineages. Furthermore, the deregulated miRNAs, whose expressions correlate with the JAK2 mutational level, may be associated with the PV phenotype. Understanding the role of deregulated miRNAs in PV should provide insight into the pathogenesis of PV and may lead to novel therapeutic strategies.