Fig. 3: Plasma sEVs released by TBI patients and rats carry potent osteogenic miRNAs, such as MiR-328a-3p and MiR-150-5p.

a Heat map presentation of relative miRNAs with ≥1.5-fold difference between rat plasma sham-sEV and TBI-sEV at 24 and 72 h post-impact. b Comparison of relative miR-219a-1-3p, miR-328a-3p, miR-139-5p, miR-150-5p, miR-218a-5p, miR-32-5p content between sham-sEV and TBI-sEV of rat plasma at 24 h post-impact by qRT-PCR (n = 3; Student’s two-sided unpaired t test). P_{(miR-219a-1-3p TBI plasma sEV vs. sham plasma sEV)} < 0.0001, P_{(miR-328a-3p TBI plasma sEV vs. sham plasma sEV)} < 0.0001, P_{(miR-139-5p TBI plasma sEV vs. sham plasma sEV)} = 0.0009, P_{(miR-150-5p TBI plasma sEV vs. sham plasma sEV)} = 0.0004, P_{(miR-218a-5p TBI plasma sEV vs. sham plasma sEV)} = 0.0278, P_{(miR-32-5p TBI plasma sEV vs. sham plasma sEV)} = 0.1044. c, d BMSCs and MC3T3-E1 cells were transfected with miR-328a-3p, miR-219a-1-3p, miR-139-5p, miR-150-5p, miR-32-5p mimics, or NC (negative control) in osteogenic medium. ALP staining and western blots were performed on day 7. β-actin served as the loading control (n = 3; one-way analysis of variance with Dunnett’s multiple comparisons test was performed). P_{(BMSC NC vs. miR-328a-3p)} = 0.0007, P_{(BMSC NC vs. miR-219a-1-3p)} = 0.9998, P_{(BMSC NC vs. miR-139-5p)} = 0.9987, P_{(BMSC NC vs. miR-150-5p)} = 0.0004, P_{(BMSC NC vs. miR-32-5p)} = 0.009, P_{(MC3T3-E1 NC vs. miR-328a-3p)} = 0.0278, P_{(MC3T3-E1 NC vs. miR-219a-1-3p)} = 0.8747, P_{(MC3T3-E1 NC vs. miR-139-5p)} = 0.7938, P_{(MC3T3-E1 NC vs. miR-150-5p)} = 0.0430, P_{(MC3T3-E1 NC vs. miR-32-5p)} = 0.5791. e, f Human BMSC were transfected with miRNAs mimics, NC and their anti-miRNAs on day 7 for ALP staining. Western blot analysis of RUNX2 expression in human BMSCs. β-actin and GAPDH serve as loading control (n = 3; One-way analysis of variance with Turkey’s multiple comparisons test was performed). P_{(NC vs. miR-328a-3p)} = 0.001, P_{(miR-328a-3p vs. Anti-NC)} = 0.0011, P_{(miR-328a-3p vs. Anti-miR-328a-3p)} = 0.0001. P_{(NC vs. miR-150-5p)} = 0.0067, P_{(miR-150-5p vs. Anti-NC)} = 0.0166, P_{(miR-150-5p vs. Anti-miR-150-5p)} = 0.0001. g RT-PCR identification of sEV miR-328a-3p and miR-150-5p in TBI patients versus healthy controls (n = 10; Student’s two-sided unpaired t test). P_{(miR-150-5p TBI vs. Control)} = 0.0002, P_{(miR-328a-3p TBI vs. Control)} < 0.0001. The quantitation result were plotted as dot plots, showing the mean ± SEM of three independent experiments. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001; ns not significant.