Fig. 2: miR-125b-5p inhibition impairs the terminal enucleation of erythroblasts in vivo.

A After intra-BM injection of miR-125b-5p inhibitors in ICR mice, qRT-PCR demonstrated the decreased expression of miR-125b-5p in BM (p = 0.0162). U6 was the loading control, and the results are expressed as the mean ± SD (n = 3). B, C Comparison of the erythroblast differentiation and enucleation rate between the miR-125b-5p inhibitor injection group and the control group (17 mice per group) on injection Day 3. Statistical analysis of the percentage of CD71⁻/TER-119⁺, CD71^low/TER-119⁺ and CD71^high/TER-119⁺ erythroblasts (B, upper panel, no statistically significant difference) and nucleated cells (LDS751⁺) in CD71⁻/TER-119⁺, CD71^low/TER-119⁺ and CD71^high/TER-119⁺ erythroblasts harvested from BM (B, lower panel, p = 0.0391, p = 6.91972E-07 and p = 7.11841E-05) and nucleated cells from PB (C, p = 0.0431 and p = 0.0023). Data are expressed as the mean ± SD (*p < 0.05, **rp < 0.01, and ***p < 0.001). D Representative images of immunofluorescence staining show cells derived from BM with or without miR-125b-5p inhibitor treatment. miR-125b-5p inhibitor treatment resulted in increased NRBCs in BM. Red indicates Ter119 staining, and blue indicates DAPI nuclear staining. The arrows indicate Ter119⁺ cells with nuclei (i.e., NRBCs). Scale bars = 25 μm. E Schematics illustrating AAV vectors that carried miR-125b-5p sponge or control. mLCR5'HS2 represents the enhancer of mouse beta globin and pHbbt1 represents the promoter of mouse beta globin. F Seven days after tail vein injection of the miR-125b-5p sponge in BALB/c mice, qRT-PCR demonstrated the decreased expression of miR-125b-5p in PB (p = 0.0001). U6 was the loading control, and the results are expressed as the mean ± SD (n = 4). G Comparison of the erythroblast enucleation rate between the miR-125b-5p sponge injection group and the control group (4 mice per group) on injection Days 0, 3 and 7. Statistical analysis of the ratio of nucleated cells (Syto62⁺) in whole red blood cells (Ter119⁺) (p = 0.0388 for Day 7) as well as TER119⁺/CD71⁺ and TER119⁺/CD71⁻ erythroblasts (p = 0.0353 and p = 0.0273 for Day 3 and Day 7 TER119⁺/CD71⁻ erythroblasts respectively) harvested from PB. Data are expressed as the mean ± SD (*p < 0.05). H Comparison of the erythroblast differentiation and enucleation rate between the miR-125b-5p sponge injection group and the control group (4 mice per group) from BM erythroid cells at Day 7 post injection. Upper panel is statistical analysis of erythroblast differentiation, significant difference was only detected in CD71⁻/TER119⁺ erythroblasts (p = 0.0022). Lower panel is statistical analysis of the ratio of nucleated cells (Syto62⁺ ) in TER119⁺/CD71^high, TER119⁺/CD71^low and TER119⁺/CD71⁻ erythroblasts harvested from BM (p = 0.0004 for TER119⁺/CD71⁻ erythroblasts). Data are expressed as the mean ± SD (***p < 0.001). I Left panel: Representative images of the bone marrow smear stained with Wright-Giemsa dye. Arrows indicate the enucleated cells. Scale bars = 50 μm. Right panel: Statistical analysis of the percentage of BM nucleated cells calculated from four random views. miR-125b-5p sponge treatment resulted in increased NRBCs in BM. (p = 0.0193) (*p < 0.05).