Fig. 7

Exosomal miR-15b-5p and miR-378a-3p promote NET formation by targeting PDK1. a Differential expression of miRNAs between PBS-Exo and LPS-Exo is expressed as fold change, as determined by miRNA-seq. b qRT-PCR verified the miRNAs identified by miRNA-seq; results are shown as fold change (n = 6). c The overexpression of selected miRNAs (miR-24-3p, miR-15b-5p, miR-25-3p, miR-126-3p, miR-378a-3p, and miR-155-5p) was analyzed to assess their effect on NET formation. The supernatant dsDNA concentration was quantified by PicoGreen fluorescent dye. d Sequence alignment between miR-15b-5p, miR-378a-3p, and their putative binding sites (in red letters) in the PDK1 3′-UTR. Mutation of the miR-15b-5p and miR-378a-3p target sites (in blue letters) is shown below. e Luciferase reporter assay was performed to detect the relative luciferase activities of WT and Mut PDK1 reporters. The Renilla luciferase vector was used as an internal control. qRT-PCR analysis of relative PDK1 mRNA levels (f) and western blot (g) of PDK1, p-Akt, Akt, p-mTOR, mTOR, and LC3B in differentiated HL-60 cells transfected with miR-15b-5p or miR-378a-3p mimics as indicated. h, i Prior to coculturing with LPS-Exo for 24 h, differentiated HL-60 cells were transfected with control, miR-15b-5p or miR-378a-3p inhibitors, or both for 24 h. h The expression levels of PDK1, p-Akt, Akt, p-mTOR, mTOR, and LC3B in differentiated HL-60 cells were measured by western blot. i Quantification of dsDNA in the supernatant of differentiated HL-60 cells using PicoGreen fluorescent dye. j–l PMNs were cocultured with PBS, PBS-Exo, or LPS-Exo for 14 h. Relative expression levels of miR-15b-5p, miR-378a-3p, and PDK1 were measured by qRT-PCR (j, k). Western blot of PDK1 in PMNs (l). Graphs represent means ± SEM, n = 3; *P < 0.05, **P < 0.01 compared within two groups