Fig. 3: miR-125b-5p expression is positively correlated with erythroblast enucleation efficiency.

A CD34⁺ hematopoietic stem/progenitor cells (HSPCs) were induced toward erythrocytes. Erythroid maturation was visualized by staining with May-Grünwald Giemsa on Days 0, 7, 10 and 14 (left panel). Scale bars = 20 μm. The expression of miR-125b-5p during erythropoiesis on Days 0, 7, 10, 12 and 14 was determined by qRT-PCR (right panel, p = 0.0008 for Day 12 and p = 0.0007 for Day 14). B miR-125b-5p was significantly upregulated in the erythroid enucleation induction of K562 (on Day 10) and TF-1 (on Day 8) cells (p = 0.0065 for K562 and p = 4.03263E-05 for TF-1). C, D miR-125b-5p expression in different erythroid developmental stages. In CD34⁺ HSPCs at erythroid induction day 14, erythroblasts were stained and sorted by CD71/CD235a (C) or CD49d (α4 integrin)/Band 3 (D) In the CD71/CD235a system (C), populations were gated as follows: P2, CD71⁺CD235a^-; P3, CD71⁺CD235a^med; P4, CD71⁺CD235a⁺; and P5, CD71⁻CD235a⁺ (representative cytospin images of the sorted populations are shown below). In a CD49d/Band 3 system (D), populations were gated as follows: P2, CD49d⁺Band 3^-; P3, CD49d⁺Band 3^med; P4, CD49d⁺Band 3⁺; and P5, CD49d^-Band 3⁺. qRT-PCR analysis (right panel) demonstrated elevated miR-125b-5p levels from P2 to P5. U6 served as the miRNA expression control. E Exogenous upregulation of miR-125b-5p in hCB-MNC-derived erythroblasts enhanced the erythroblast enucleation rate. Overexpression of miR-125b-5p was analyzed by qRT-PCR (left panel, p = 7.82165E-06). Representative images of flow cytometry analysis show the enucleation efficiency (CD235⁺SYTO 16^-, center panel). Statistical analysis of the enucleation rates from five independent experiments is expressed as the mean ± SD (right panel; p = 0.0006). F Representative images of the enucleation morphology on stained cytospins are shown. Red arrows indicate enucleated cells, and black arrows indicate nucleated cells. Scale bars = 20 μm. G Exogenous downregulation of miR-125b-5p in erythroblasts reduced their enucleation rate. Left panel shows miR-125b-5p levels after inhibitor transfection (p = 0.0012). Middle panel shows representative flow cytometry analysis of erythroblast enucleation. Right panel shows statistical enucleation rate (p = 0.0114). Statistical analysis was based on data from five independent experiments. H Stable miR-125b-5p overexpression improved K562 erythroid cell enucleation. The expression of miR-125b-5p was detected with qRT-PCR (left panel, p = 0.0008). Overexpression of miR-125b-5p by the pcDNA3.1 vector in K562 cells increased the percentage of CD235a⁺LDS751⁻ populations. Statistical analysis of three independent experiments is expressed as the mean ± SD (right panel) (p = 0.0135). In qRT-PCR analyses, U6 was the loading control in (A–D) and (H), while cell number was chosen as the loading control in (E) and (G), and the results are expressed as the mean ± SD (n = 3; *p < 0.05, **p < 0.01, and ***p < 0.001).