Fig. 1: Abnormal expression of miR-125b-5p and its correlation with increased nucleated RBCs in patients with polycythemia vera.

A Quantitative RT-PCR shows endogenous miR-125b-5p expression in MNCs isolated from the PB of healthy controls and patients with PV (n = 3). Relative expression was normalized to U6 miRNA and is expressed as the mean ± SD (p = 0.0008). B Flow cytometry analysis shows the NRBC percentage in the PB of healthy controls or patients with PV. C qRT-PCR of miR-125b-5p expression from three independent experiments with healthy RBCs or RBCs from patients with PV. The results were normalized with cell number (p = 0.0309). D Representative images of the flow cytometry analysis of nucleated (LDS751⁺) CD235a⁺CD71⁻ erythrocytes (NRBCs) in the BM of healthy controls and patients with PV. E qRT-PCR using the indicated cells isolated from the BM of healthy controls and patients with PV shows the expression trend of miR-125b-5p during erythropoiesis (MNCs, erythroblasts and NRBCs; **p = 0.0058). F Schematic illustration representing the erythrocyte induction procedure and transfection with the miR-125b-5p mimic. G Representative images of flow cytometry analysis of CD71 and CD235a expression at erythroblast differentiation Day 14 on BM-MNCs from healthy controls and patients with PV. On enucleation Day 6, qRT-PCR (p = 0.0007) shows miR-125b-5p expression (H), and representative images show the percentage of enucleated erythroblasts (CD235a⁺/SYTO 16⁻) from healthy controls and patients with PV (I) The right panel of (I) shows the statistical analysis of data from four independent flow cytometry analyses, and the bar graph represents the mean ± SD (p = 0.0016). J qRT-PCR shows the overexpression of miR-125b-5p (p = 3.09489E-06) and representative images of flow cytometry analysis (K) illustrate the enucleation status of PV erythroblasts after treatment with miR-125b-5p mimic or control miRNA. The right panel of (K) shows the statistical analysis of data from four independent flow cytometry analyses, and the bar graph represents the mean ± SD (p = 0.0032; *p < 0.05, **p < 0.01, and ***p < 0.001).