Fig 2: circITGB1 acts as a miR-342-3p sponge to regulate NFAM1 expression in mdDCs. (a) Prediction of miRNA binding sites in the circITGB1 sequence. (b) Luciferase reporter assay for the luciferase activity of circITGB1 or circITGB1-mut 293T cells cotransfected with five miRNA mimics or miR-control. Data are the means ± SD of three independent experiments. ∗∗P < 0.01 vs. miR-control+circITGB1 mut. (c, d) qPCR detected miR-342-3p (c) or circITGB1 (d) enriched from human mdDC lysates by biotinylated-oligonucleotide probe for circITGB1 or miR-342-3p. (e) Colocalization between miR-342-3p and circITGB1 was detected by RNA in situ hybridization. (f) The effect of circITGB1 knockdown on miR-342-3p expression was investigated by qPCR. (g) Predicted NFAM1 target region and miR-342-3p through TargetScan website. (h) Luciferase assay in 293A cells cotransfected with the indicated RNA or luciferase reporters. ∗∗P < 0.01 vs. miR-control+NFAM1 3′UTR mut. (i) Western blotting analyzed NFAM1 expression; β-actin was used as the loading control. (j) Western blotting analyzed NFAM1 expression in mdDCs transfected with the indicated RNA; β-actin was used as the loading control. (k) Western blotting analyzed NFAM1 expression; β-actin was used as the loading control. Significance was analyzed using Student's t-test or one-way ANOVA. ∗P < 0.05, ∗∗P < 0.01.

Fig 3: Model of EV-circITGB1 regulates maturation of dendritic cells (DCs). EV-circITGB1 acts as a miR-342-3p sponge to regulate NFAM1 expression, which adjusts the expression of DC maturation markers CD80 and CD86.

Fig 4: EV-circITGB1 regulates mdDC maturation via miR-342-3p and NFAM1. (a) Human mdDCs were pretreated with circITGB1-ir, circITGB1, miR-control, miR-342-3p, NFAM1-sc, or NFAM1-siRNA for 1 h prior to overnight stimulation with LPS (100 ng/mL). Cells were collected, stained, and analyzed for surface marker expression using flow cytometry. Bar graphs show expression of CD80 and CD86 on viable CD11c+ cells in 4 donors. Median fluorescence intensity of gated cells for corresponding surface markers ± SD, from 4 independent cultures. (b) Human mdDCs were pretreated with control siRNA, circITGB1 siRNA, anti-miR-control, anti-miR-342-3p, or DMA (21.3 mM) for 1 h prior to overnight stimulation with LPS (100 ng/mL). Cells were collected, stained, and analyzed for surface marker expression using flow cytometry. Bar graphs show expression of CD80 and CD86 on viable CD11c+ cells and are representative of 4 donors. Median fluorescence intensity of gated cells for corresponding surface markers ± SD, from 4 independent cultures. ∗∗P < 0.01.