Regulated expression of microRNAs in normal and polycythemia vera erythropoiesis

Abstract

Objective: Polycythemia vera (PV) is a myeloproliferative disorder, arising from the acquired mutation(s) of a hematopoietic stem cell. The JAK2 V617F somatic mutation is found in most PV patients; however, it is not the disease-initiating mutation. Because microRNAs (miRNAs) play a regulatory role in hematopoiesis, we studied miRNA expressions in PV and normal erythropoiesis.

Methods: Peripheral blood mononuclear cells were cultured in a three-phase liquid system resulting in synchronized expansion of erythroid progenitors. Using gene-expression profiling by CombiMatrix MicroRNArray, we searched for PV-specific changes at days 1, 14, and 21. Twelve miRNA candidates were then reevaluated by quantitative real-time polymerase chain reaction in a larger number of samples obtained from progenitors at the same stage of differentiation.

Results: A significant difference in miR-150 expression was found in PV. In normal erythropoiesis, three expression patterns of miRNAs were observed: progressive downregulation of miR-150, miR-155, miR-221, miR-222; upregulation of miR-451, miR-16 at late stages of erythropoiesis; and biphasic regulation of miR-339, miR-378. The miR-451 appears to be erythroid-specific.

Conclusions: We identified the miRNAs with regulated expression in erythropoiesis; one appeared to be PV-specific. Their miRNA expression levels define early, intermediate, and late stages of erythroid differentiation. The validity of our findings was confirmed in nonexpanded peripheral blood cells.

Figure 1. Induction of erythroid differentiation from peripheral blood MNCs

A) Morphology analysis of Wright-Giemsa stained cells at selected days (magnification 100×). The representative populations are shown: day7 – immature early erythroid progenitors, day11 - proerythroblasts and basophilic erythroblasts, day14 - mostly basophilic erythroblasts, day16 - basophilic and polychromatophilic erythroblasts, day21 – late normoblasts and some reticulocytes; B) Flow cytometry analysis of surface antigens, CD71 and CD235a, on expanded cells at different stages of differentiation. The cells were immunostained with (FITC)-conjugated anti-CD71 and (PE)-conjugated anti-CD235a antibodies. In the plot, X- and Y-axes indicate the relative fluorescence of PE and FITC, respectively. The gated regions define characteristic expression patterns of the surface antigens: CD71^med and CD235a^low (R1), - CD71^high and CD235a^low (R2), - CD71^high and CD235a^high (R3), - CD71^med and CD235a^high (R4), - CD71^low and CD235a^high (R5).

Figure 2. Expression of the differentially regulated miRNAs during erythroid differentiation

The one-way ANOVA analysis (P<0.01) selected 30 miRNAs which were differentially expressed between the 3 groups (Day1 versus Day14 versus Day21). The control samples from day21 were pooled because of limited amount of RNA. The relative gene expressions are expressed by a gradient intensity of color, as shown in the grey scale at the top. The darkest color indicates no expression and the lightest color indicates maximal expression. C-control, PV- polycythemia vera patient.

Figure 3. Gene expression patterns of the miRNAs depicting increased levels during erythroid differentiation

Gene expression was determined by qRT-PCR at the defined time points. Relative gene expression was calculated based on generation of standard curve and normalized against the endogenous control RNAU6B. The plotted data (arbitrary units) express as mean with standard error. Statistical significance between control and PV cells was calculated by t-test at each time point (*P<0.05, **P<0.01). Control - healthy controls, PV- polycythemia vera patients, D – day.

Figure 4. Gene expression patterns of the miRNAs depicting decreased levels during erythroid differentiation

Gene expression was determined by qRT-PCR at the defined time points. Relative gene expression was calculated based on generation of standard curve and normalized against the endogenous control RNAU6B. The plotted data (arbitrary units) express as mean with standard error. Statistical significance between control and PV cells was calculated by t-test at each time point (*P<0.05, **P<0.01). Control - healthy controls, PV- polycythemia vera patients, D – day.

Figure 5. Gene expression patterns of the miRNAs depicting biphasic regulation during erythroid differentiation

Gene expression was determined by qRT-PCR at the defined time points. Relative gene expression was calculated based on generation of standard curve and normalized against the endogenous control RNAU6B. The plotted data (arbitrary units) express as mean with standard error. Statistical significance between control and PV cells was calculated by t-test at each time point (*P<0.05, **P<0.01). Control - healthy controls, PV- polycythemia vera patients, D – day.

Figure 6. Expression levels of the miRNAs in non-expanded peripheral blood cells from controls and PV patients

Gene expression was determined by qRT-PCR. Relative gene expression was calculated based on generation of standard curve and normalized against the endogenous control RNAU6B. The plotted data (arbitrary units) express as mean with standard error. Statistical significance between control and PV cells was calculated by t-test in each cell lineage (*P<0.05, **P<0.01). Control - healthy controls, PV - polycythemia vera patients, MNC - mononuclear cells, GRAN - granulocytes, RETIC - reticulocytes, PLAT - platelets.

Figure 6. Expression levels of the miRNAs in non-expanded peripheral blood cells from controls and PV patients

Gene expression was determined by qRT-PCR. Relative gene expression was calculated based on generation of standard curve and normalized against the endogenous control RNAU6B. The plotted data (arbitrary units) express as mean with standard error. Statistical significance between control and PV cells was calculated by t-test in each cell lineage (*P<0.05, **P<0.01). Control - healthy controls, PV - polycythemia vera patients, MNC - mononuclear cells, GRAN - granulocytes, RETIC - reticulocytes, PLAT - platelets.