Differential Regulation of MicroRNA Expression In Polycythemia Vera CD34+ Cells

Polycythemia vera (PV) is a clonal stem cell disorder characterized by unregulated red cell, white cell and platelet production, a phenotype attributable to an activating point mutation (V617F) in JAK2, a tyrosine kinase utilized for signaling by Type 1 hematopoietic growth factor receptors. However, it is generally conceded that JAK2 V617F is not the initiating mutation in PV, nor are any of the other gene mutations, deletions and amplifications discovered to date. Deregulated gene transcription is also a feature of PV but a unified mechanism for this also cannot be ascribed to the currently known genetic defects. MicroRNAs (miRs) are non-coding 18–22nt RNAs that function as posttranscriptional gene repressors by binding to complementary target sequences in the mRNA 3′UTR to inhibit protein translation. There is growing evidence that miRs regulate hematopoiesis not only at the committed hematopoietic progenitor cell level but also at the level of the hematopoietic stem cell (HSC). Studies of miR behavior in PV have documented differential expression during erythroid differentiation as well as differential expression in granulocytes and platelets. However, PV is a stem cell disorder and to date there has been no analysis of miR behavior in PV HSC. We report here differential miR expression in PV peripheral blood (pb) CD34+ cells.

We studied pb CD34⁺ cells from eight PV patients (four females and four males, all JAK2 V617F-positive) and six normal donors (three females and three males) in conjunction with a study of mRNA expression in PV pb CD34+ cells by oligonucleotide microarray. Using total cell RNA, microRNA expression was analyzed with the Agilent Human microRNA Microarray Kit Version 3, which contains probes for 866 human miRs. The TaqMan MicroRNA Assay (Applied Biosystems) was used for quantitative reverse-transcription PCR to verify differential miR expression. The Student unpaired t test was used for statistical analysis and P values £ 0.05 were considered significant.

Overall, in the 8 PV patients as a group, there were 130 positive calls with differential regulation of 81 miRs (p <0.05) (23 up regulated and 58 down regulated) and there was no correlation between miR expression and the JAK2 V617F allele burden. Given the phenotypic and genotypic differences between men and women PV patients, we analyzed differential miR expression separately by gender to identify gender-specific effects. 75 miRs (26 up and 49 down regulated) were differentially expressed in the male PV patients and 32 (7 up and 25 down regulated) in the women, confirming similar observations of gender-related differences in global mRNA expression in PV pb CD34+ cells. Amongst the differentially regulated miRs in the group as a whole were the miR-H1 group, miR-125a-3p and 196b. The miR-H1 group, also known as miR-17-92, is an oncomir with a unique gene structure from which one primary transcript yields six distinct miRs. This cluster was only up regulated in male PV patients. Likewise, miR-125a-3p was also up regulated only in males. Its expression is dependent on DICER, a gene also only up regulated in male patients. MiR-125a-3p has been identified as being preferentially expressed in HSC, acts to expand HSCs by preventing apoptosis and favors a myeloid fate. MiR-196b, which was concordantly down regulated in both men and women, is normally up regulated in HSC. Altogether, consistent with their different phenotypes, 31 miRs were differently regulated solely in the male patients (16 up regulated and 15 down), while only 12 miRs were differently regulated solely in the women (2 up and 10 down regulated) Seven miRs were concordantly up (miR-575) or down regulated (miR-150; miR-181c; miR-196b; miR-551b; miR-769-5p and miR-874) by both the men and women patients. Notably, down regulation of miR-150 at the HSC level is consistent with its role in lineage-specific progenitor cell commitment. Since there appears to be little overlap with respect to target genes for this core group of miRs, multiple venues for translational control are likely to be involved in the pathogenesis of PV.

These data establish the presence of differential miR expression in PV HSC and provide insight into potential mechanisms for PV HSC expansion, confirm a genetic basis for the gender-specific differences that characterize PV and identify novel therapeutic targets for drug discovery.

No relevant conflicts of interest to declare.