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. 2024 Jul 4;36:508–523. doi: 10.1016/j.bioactmat.2024.06.035

Fig. 3.

Fig. 3

BMM-EV miR-378a-3p and miR-140-5p may coordinately regulate SSPC lineage fate.

(A) miRNA microarray analyzes the differently expressed microRNAs in lean BMM-EVs and obese BMM-EVs. The red rectangle circles three up-regulated microRNAs in obese BMM-EVs, and the blue rectangle circles three down-regulated microRNAs. The arrows mark the microRNAs chosen for further functional verification. n = 3 per group. (B) qRT-PCR analysis of the relative expression levels of osteogenic genes (Alp, Sp7) and adipogenic genes (Ppar-g, Fabp4) of the SSPCs transfected with three up-regulated microRNAs (miR-140-5p, miR-221-5p, and miR-1839-5p) and their negative controls, which culture in osteogenic and adipogenic medium for 6 days, respectively. NC, negative control. n = 3 per group. (C) qRT-PCR analysis of the relative expression levels of osteogenic genes (Alp, Sp7) and adipogenic genes (Ppar-g, Fabp4) of the SSPCs transfected with three downregulated microRNAs (miR-378a-3p, miR-378c, and miR-378d) and their negative controls for 6 days, respectively. n = 3 per group. (D) Representative images of Alizarin red staining (D, top) and oil red O staining (D, bottom) of the SSPCs transfected with miR-140 and negative controls. Scale bar: 100 μm. (E) Representative images of Alizarin red staining (E, top) and oil red O staining (E, bottom) of the SSPCs transfected with miR-378a and negative controls. Scale bar: 100 μm. (F-I) BMM-EVs overloaded with miR-140 and miR-378a regulate SSPC lineage fate between osteoblasts and adipocytes. qRT-PCR analysis of the relative expression levels of osteogenic genes (F and H) and adipogenic genes (G and I) in miRNA-140 enriched macrophage-derived extracellular vesicle and miR-378a enriched macrophage-derived extracellular vesicle-treated SSPCs. n = 3 per group. Data is shown as mean ± SD. *P < 0.05, **P < 0.01, ns, no significant. (Welch's test is used in the Fabp4 expression of Fig. 3B, Ppar-g and Fabp4 expression of Fig. 3C, and Alp, Sp7 expression in Fig. 3H, non-parametric test is used in Sp7 expression of Fig. 3C and student t-test is used in others).