Fig. 4: Exosomal miR-19a targets PTEN/AKT pathway to promote osteoclastogenesis.

a Exosomes were labeled with GFP by ectopic expression of PalmGFP in MCF7BoM2 cells. OC cells were then treated with the labeled exosomes. After 24 h, uptake of exosome by osteoclast cells was monitored by confocal microscope. Scale bar, 10 µm. b Luciferase reporter assay for the constructs containing wild-type PTEN 3′UTR or mutant PTEN 3′UTR was performed after transfecting the cells with the miR-19a expression vector. p = 0.0001 (WT + PCDH vs WT + PCDH-miR-19a, two-sided student’s t-test). Data are presented as mean values ± SEM. c Taqman PCR for miR-19a expression (p = 0.0131, n = 3), and SYBR Green qPCR for PTEN expression (p = 0.0232, n = 3) in RAW264.7/GFP and RAW264.7/miR-19a were performed. Two-sided student’s t-tests were performed. Data are presented as mean values ± SEM. d Western blot analysis for PTEN, phospho-NF-κB P65, total NF-κB P65, phospho-AKT and total AKT expression in RAW264.7/GFP and RAW264.7/miR-19a cell lines. The band intensity was quantified and normalized to that of control (left panels). e TRAP staining of RAW264.7/GFP and RAW264.7/miR-19a cells. On day 4, the number of differentiated osteoclasts (p = 0.0080, n = 5 vs 6) and the size of these cells (p < 0.0001, n = 30 vs 40) were measured. Two-sided student’s t-tests were performed. Data are presented as mean values ± SEM. Scale bar, 100 µm. f Mouse BMMs were induced into OC cells with the treatment of exosomes and cytokines. Taqman PCR for miR-19a expression (p = 0.0008, n = 3) and SYBR Green qPCR for PTEN (p = 0.0271, n = 3) expression were performed for OC cells treated with exosomes from MCF7 and MCF7BoM2. Two-sided student’s t-tests were performed. Data are presented as mean values ± SEM. g Western blot analysis for PTEN, phospho-NF-κB P65, total NF-κB P65, phospho-AKT and total AKT expression in mouse BMMs treated with exosomes from MCF7 and MCF7BoM2. The band intensity was quantified and normalized to that of control (left panels). h TRAP staining was performed for mouse BMMs treated with exosomes from MCF7 and MCF7BoM2. On day 4, the number of differentiated osteoclast (p = 0.000036, n = 5 vs 5) and their sizes (p = 0.0323, n = 10 vs 23) were measured. Two-sided student’s t-tests were performed. Data are presented as mean values ± SEM. Scale bar, 100 µm. i Bone-resorbing activities of OC treated with exosomes from MCF7 and MCF7BoM2 were measured by the bone-resorption assay. BMMs were seeded on a bone chip and treated with exosomes and cytokines. Resorption pits were measured and compared (p = 0.0070, n = 4). Two-sided student’s t-tests were performed. Data are presented as mean values ± SEM. j Taqman PCR for miR-19a expression (p = 0.0291, n = 3) and SYBR Green qPCR for PTEN expression (p = 0.0047, n = 3) were performed for mouse BMMs treated with exosomes from MCF7BoM2/miR-19aKO and MCF7BoM2/ScrambledKO. Two-sided student’s t-tests were performed. Data are presented as mean values ± SEM. k Western blot analysis was performed for PTEN, phospho-NF-κB P65, total NF-κB P65, phospho-AKT, and total AKT expression in mouse BMMs treated with exosomes from MCF7BoM2/miR-19aKO and MCF7BoM2/ScrambledKO. The band intensity was quantified and normalized to that of control (left panels). l TRAP staining was performed for mouse BMMs that were treated with exosomes from MCF7BoM2/miR-19aKO and MCF7BoM2 /ScrambledKO. On day 4, the number of differentiated osteoclast cells (p = 0.0005, n = 5 vs 5) and their sizes were measured (p = 0.0029, n = 11 vs 38). Scale bar, 100 µm. Two-sided student’s t-tests were performed. Data are presented as mean values ± SEM. m The bone-resorbing activity of OC was measured by the bone-resorption assay using BMMs that were treated with exosomes from MCF7BoM2/miR-19aKO and MCF7BoM2/ScrambledKO (p = 0.0025, n = 24 vs 20). Two-sided student’s t-tests were performed. Data are presented as mean values ± SEM.