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Figure 5. MSC-EVs-derived miR-21a-5p is functionally associated with OA progression and macrophage regulation. (A) Illustration of the miRNA sequencing of BMSC-derived MSC-EVs; (B) Relative proportion of miRNAs in total miRNA reads. The top 10 most abundant miRNAs in BMSC-derived MSC-EVs identified by miRNA sequencing, ranked according to normalized sequencing read abundance; (C) High expression of the top 10 miRNAs in MSC-EVs was confirmed by RT-qPCR; (D) RT-qPCR analysis of the expression of the top 10 miRNAs in macrophages stimulated with MSC-EVs for 24 h (n = 3 independent biological experiments); (E) RT-qPCR analysis of top 10 miRNAs expression in macrophages stimulated with LPS for 24 h (n = 3 independent biological experiments); (F) RT-qPCR analysis of the expression of miR-21a-5p in SF-EVs (n = 5 independent human donors per group); (G) SOFG staining of the cartilage from OA patients. Scale bars: 20 μm; (H) Representative images and quantitation results of FISH for miR-21a-5p in human OA synovial tissue (n = 5 independent human donors per group); (I) Illustration of the effect of BMSC-derived MSC-EVs on the expression of miR-21a-5p in macrophages; (J) RT-qPCR analysis of the expression of miR-21a-5p in the co-cultured macrophages (n = 3 independent biological experiments); (K and L) Representative images and quantitation results of FISH for miR-21a-5p in mouse knee synovial tissue (n = 3); (M and N) Representative images and quantitation results of FISH for miR-21a-5p in mouse subchondral bone marrow cavity (n = 3 independent biological experiments). Student’s t-test was used in (D-H). One-way ANOVA followed by Tukey’s multiple-comparison test was used for comparison in multiple groups (J), (L), and (N). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001. (A) and (I) were created with BioRender.com. MSC-EVs: Mesenchymal stem cell-derived extracellular vesicles; BMSCs: bone marrow mesenchymal stem cells; miRNA: microRNA; RT-qPCR: quantitative real-time polymerase chain reaction; LPS: lipopolysaccharide; SF-EVs: synovial fluid-derived extracellular vesicles; OA: osteoarthritis; SOFG: Safranin O-Fast Green; FISH: fluorescence in situ hybridization; ANOVA: analysis of variance.