Fig. 3
Human GA-MSCs-derived exosomal miR-21 upregulated CD73 expression on MDSCs through the PTEN/PI3K/AKT/HIF-1α pathway
(A) CD73 expression in hu-BM-MSCs-EXOs (exosomes derived from human BM-MSCs) and hu-GA-MSCs-EXOs (exosomes derived from human GA-MSCs) was measured using western blotting. PBMC-derived exosomes were used as a negative control. Quantification of the fold change in the CD73/β-actin ratio (normalized to hu-BM-MSCs-EXOs) is shown. (B, C) Exosomes derived from human GA-MSCs with or without DICER knockdown were used to stimulate human PBMCs. The percentage of CD33+HLA-DR− MDSCs and the expression of CD73 on MDSCs were measured by flow cytometry. (D) Distribution of the top 10 most highly expressed miRNAs in human GA-MSCs-derived exosomes. (E) The ratio of GA-MSCs-derived exosome intensity versus BM-MSCs-derived exosome intensity is presented for the top 10 most highly expressed miRNAs. (F) The upregulated miRNAs in the top 10 most highly expressed miRNAs were transfected into glioma patient-derived CD33+HLA-DR− MDSCs. The expression of CD73 was measured using qRT‒PCR. (G, H) PBMCs were stimulated with human GA-MSCs-derived exosomes and transfected with miR-21 inhibitor. The percentage of CD33+HLA-DR− MDSCs and the expression of CD73 on MDSCs were measured by flow cytometry. (I) MDSCs obtained by stimulating CD33+ cells with GA-MSCs-derived exosomes were transfected with miR-21 inhibitor. CD33-depleted PBMCs were labeled with CFSE and cocultured with MDSCs for 72 h. CD8+ T-cell proliferation was measured by flow cytometry (J, K). The percentage of MDSCs and CD73 expression on MDSCs induced by miR-NC or miR-21 mimics and nonsense sequence or PTEN overexpression plasmids were measured using flow cytometry. (L) The expression of HIF-1α, CD73, p-AKT and PTEN was measured in miR-21-overexpressing MDSCs transfected with PTEN overexpression plasmid or treated with a PI3K inhibitor (LY294002) or HIF-1α inhibitor (2-ME2). Quantification of the fold change in the HIF-1α/β-actin, CD73/β-actin, p-AKT/β-actin and PTEN/β-actin ratios (normalized to Ctrl) are shown. The data are presented as the mean ± SD; *p < 0.05, **p < 0.01, and ***p < 0.001