Fig 2: Osteoclast-secreted miR-324 silences ARHGAP1 during osteogenic differentiation(A) Relative luciferase activity of reporter containing the 3' UTR of ARHGAP1 in MSCs upon culturing with OC-sEVs, miR-NC-sEVs, miR-324-sEVs, and miR-324-sEVs + miR-324 inhibitor; n = 5. (B) Relative mRNA expression levels of Col1a1, Alpl, Sp7, and Runx2 in MSCs cultured with OC-sEVs, miR-NC-sEVs, miR-324-sEVs, miR-324-sEVs + annexin V, miR-324-sEVs + miR-324 inhibitor, and miR-324-sEVs + ARHGAP1; n = 3. (C) Relative expression levels of ARHGAP1 in indicated groups; n = 3. (D) Western blot analysis of ARHGAP1, RhoA, ROCK, p-MYPT1, and ß-actin expression in MSCs cultured with OC-sEVs, miR-NC-sEVs, miR-324-sEVs, miR-324-sEVs + annexin V, miR-324-sEVs + miR-324 inhibitor, and miR-324-sEVs + ARHGAP1; n = 3. (E) Cell viability evaluation of MSCs in indicated groups at 0, 1, 3, 5 and 7 days; n = 3. (F) Representative images of alizarin red staining of MSCs cultured with OC-sEVs, miR-NC-sEVs, miR-324-sEVs, miR-324-sEVs + annexin V, miR-324-sEVs + miR-324 inhibitor, and miR-324-sEVs + ARHGAP1. Scale bar represents 100 µm. (G) Quantification analysis of calcium deposit of MSCs in indicated groups; n = 5. The data in the figures represent the averages ± SD. *p < 0.05, **p < 0.01, for differences between the treatment and control groups.

Fig 3: sEV-miR-324 derived from osteoclasts can be delivered into MSCs and induce osteogenic differentiation(A) The expression profile of miRNAs in sEVs secreted during osteoclast differentiation. sEVs from BMMs were used as a normalization control. Red color represents higher expression, and blue color represents lower expression relative to the control. (B) ALP activity assay shows that overexpression of miR-324 promoted osteogenic differentiation; n = 3. (C and D) Relative expression levels of (C) miR-324 and (D) pri-miR-324 in MSCs cultured with sEVs from miR-324 overexpression (miR-324-sEVs) or knockdown (anti-miR-324-sEVs) osteoclasts; n = 3. (E) Cell viability evaluation of MSCs cultured with miR-324-sEVs or anti-miR-324-sEVs using a CCK-8 test at 1, 3, 5, and 7 days; n = 5. (F) Relative mRNA expression levels of Col1a1, Alpl, Sp7, and Runx2 in MSCs cultured with miR-324-sEVs or anti-miR-324-sEVs; n = 3. (G) Western blot analysis of COL1A1, RUNX2, ALP, and ß-actin in MSCs cultured with miR-324-sEVs or anti-miR-324-sEVs. (H) Representative images of alizarin red staining of MSCs cultured with miR-324-sEVs or anti-miR-324-sEVs. Scale bar represents 100 µm. (I) Quantification analysis of calcium deposit of MSCs in indicated groups; n = 5. Data in the figures represent the averages ± SD. *p < 0.05, **p < 0.01, for differences between the treatment and control groups.

Fig 4: MiR-144-5p impairs BMSCs osteogenesis differentiation via targeting Smad1. a Venn diagram of the targeting genes of miR-144-5p predicted by microT, TargetScan, miRDB, and miRmap. b–d Western blotting and qRT-PCR analysis of Smad1 protein and mRNA levels, respectively, in BMSCs transfected with mimic-NC, miR-144-5p-mimic, inhibitor-NC or miR-144-5p-inhibitor e WT and mutated miR-144-5p recognition sites within the Smad1 3’ UTR were constructed for the dual-luciferase reporter assay and the results demonstrated Smad1 as a direct target gene for miR-144-5p. f–h After BMSCs were con-transfected with miR-144-5p-inhibitor or si-Smad1; the mRNAs and proteins of Smad1, RUNX2, ALP, collagen ? and OCN were determined using qRT-PCR and Western blotting. I the mineralization in BMSCs following different treatments was measured via Alizarin red staining and ALP staining. j–k The statistical data of Alizarin red staining and ALP staining. Data are presented as the mean±SD, and all experiments were repeated three times. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001

Fig 5: dBMDM-exos hinder BMSCs osteogenesis differentiation in vitro. a–c The protein and mRNA levels of some osteogenic genes in BMSCs treated with PBS, nBMDM-exos or dBMDM-exos were determined by Western blotting and RT-qPCR analyses, respectively. d The proportion of mineralization in treated BMSCs was shown by Alizarin red staining and ALP staining assays. e, f The Alizarin red and ALP staining were quantitatively determined. Data are presented as the mean ± SD (n = 3 per group), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001