CircN4bp1 Facilitates Sepsis-Induced Acute Respiratory Distress Syndrome through Mediating Macrophage Polarization via the miR-138-5p/EZH2 Axis

Figure 4

CircN4bp1 acts as a miRNA sponge for miR-138-5p. (a) Schematic showing the predicted miR-138-5p sites in circN4bp1. (b) qRT-PCR quantification of miR-138-5pin the serum and monocytes of 40 ARDS patients postsepsis and 40 healthy controls, and data are expressed as ( vs. serum of healthy controls; # vs. monocytes of healthy controls). (c) qRT-PCR quantification of miR-138-5p in the two ex vivo macrophage cell lines transfected withcircN4bp1 lentivirus plasmids (circN4bp1-OE) or scrambled control (NC), and data are expressed as ( vs. scrambled control of raw264.7 cells; # vs. scrambled control of MH-S cells). (d) Luciferase assays in raw264.7 cells cotransfected with a scrambled control, miR-138-5p mimic, and a luciferase reporter plasmid containing wild-type circN4bp1 (circN4bp1-WT) or muted-type circN4bp1 (circN4bp1-Mut). ( vs. circNC group; # vs. circN4bp1-Mut group). (e) RIP assay was performed using input, IgG, or anti-Ago2 antibodies. Relative expression of miR-138-5p assayed by qRT-PCR. (f) RIP assay and relative expression of circN4bp1 determined by qRT-PCR. CircANRIL (a circular RNA reported not to bind to AGO2) was used as a negative control. Data of three independent assays. . RAW264.7 and MH-S were transfected with miR-138-5p mimic or inhibitor and then exposed to either LPS (50 ng/ml) or IL-4 (10 ng/ml) for an additional 24 h. (g) Representative Western blot depicting raw264.7 cell lysates probed for iNOS, Arg-1, and GAPDH. Expression levels of iNOS and Arg-1 were quantified by densitometry and normalized using GAPDH. (h) Representative western blot depicting MH-S cell lysates probed for iNOS, Arg-1, and GAPDH. Expression levels of iNOS and Arg-1 were quantified by densitometry and normalized using GAPDH. ( vs. NC group, # vs. LPS stimulated group, ^@ vs. miR-138-5p mimic +LPS group, & vs. IL-4 stimulated group, and % vs. miR-138-5p mimic +IL-4 group determined by one-way ANOVA for multiple group comparisons).

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