Fig. 5: The upregulation of miR-125b-5p is highly correlated with actin rearrangement, nuclear condensation and mitochondrial clearance during the final stage of erythropoiesis.

A–J Immunofluorescence staining and confocal microscopy indicating the status of actin (phalloidin, red), cytoplasm/CFSE and tubulin (green) and nuclei (DAPI, blue) in cells at the indicated induction times with or without miR-125b-5p treatment. Five random fluorescence images from each group were acquired and analyzed by HCS STUDIO software. Data from three independent experiments were statistically analyzed and are expressed as the mean ± SD. A hCB-MNC-derived erythroblasts on Day 4. Scale bars = 20 μm. B K562 cells after first-stage erythrocyte induction. Scale bars = 50 μm. C–F Overexpression of miR-125b-5p enhanced cytoskeletal remodeling. The average fluorescence area (C, p = 0.0329) and intensity (D, p = 0.0247) of phalloidin staining indicated actin rearrangement in hCB-MNC-derived erythroblasts. The average fluorescence area (E, p = 0.0056) and intensity (F, p = 0.0063) of phalloidin staining indicated actin rearrangement in K562 erythroid cells. G–J Cells with miR-125b-5p overexpression exhibited smaller nuclei. G Mean nuclear areas of hCB-MNC-derived erythroblasts (p = 0.0239). H Mean cell areas of hCB-MNC-derived erythroblasts. No significant difference was detected between the miR-125b-5p overexpression and control groups (p = 0.7225). I Mean nuclear areas of K562 erythroid cells (p = 0.0004). J Mean cell areas of K562 cells. Compared to control cells, miR-125b-5p-overexpressing cells exhibited a smaller cell size (p = 0.0045). K–O Seven days post AAV-miR-125b-sponge infection, immunofluorescence staining and statistical analysis indicated the alteration of nuclear shrinkage, cell size and the status of actin in mouse bone marrow erythroblasts. K Mean nuclear areas of BM erythroblasts, cells with miR-125b-5p KD exhibited larger nuclei. (p = 0.0112). L Mean cytoplasm areas of BM erythroblasts, cells with miR-125b-5p KD exhibited larger cytoplasm. (p = 0.0075) M Nuclear/ cytoplasmic ratio of BM erythroblasts, no statistically significant difference was detected (p = 0.1235). N, O miR-125b-5p KD affected cytoskeletal remodeling. The average fluorescence area (N, p = 0.0128) and intensity (O, p = 0.2476) of phalloidin staining indicated reduced actin aggregation after miR-125b-5p KD. P Representative images of electron micrographs of hCB-MNC-derived erythroblasts at enucleation Day 4 (left panel). The arrows indicate mitochondria. Scale bars=500 nm. miR-125b-5p overexpression destroyed mitochondrial structure and reduced the number of mitochondria. Statistical analysis of the mitochondrial number/cell from five random fields, and the results are expressed as the mean ± SD (right panel; p = 0.0021, **p < 0.01 and ***p < 0.001).