Fig. 4

circNOX4 upregulates FAP expression by sponging miR-329-5p in CAFs. A FISH was performed to evaluate the sublocation of circNOX4 (red) in CAFs and NFs, respectively. The nucleus (blue) was stained with DAPI. Scale bar = 10 μm. B Expression of circNOX4 in either the nucleus or cytoplasm of CAFs. U6 and GAPDH were used as nuclear and cytoplasmic localization markers, respectively. C Schematic illustration of the potential target miRNAs of circNOX4 predicted by miRanda and RNAhybrid. D Venn analysis of the potential binding miRNAs of circNOX4 and FAP, predicted by miRanda, RNAhybrid, and TargetScan. E The expression of miR-329-5p in CAFs and NFs was analyzed by RT-qPCR. F The effect of circNOX4 siRNAs on the expression of miR-329-5p in CAFs. G Diagram of the secondary structure of circNOX4 and the possible binding sites with miR-329-5p predicted by RNAalifold. H The sequence alignment of circNOX4-WT and circNOX4-MUT in luciferase reporter assay for validating the binding of circNOX4 and miR-329-5p. Luciferase reporter assay showing the luciferase activity of the circNOX4 luciferase reporter plasmid (WT or MUT) following transfection with miR-NC or miR-329-5p mimic into CAFs. I Ago2-RIP assay was applied to detect the expression of circNOX4 and miR-329-5p in CAFs. J RNA-FISH was performed to evaluate the location of circNOX4 (red) and miR-329-5p (green) in CAFs. The nucleus (blue) was stained with DAPI. Scale bar = 10 μm. K The potential possibility of FAP binding to miR-329-5p was predicted by RNA–Protein Interaction Prediction (RPISeq) website. The interaction probabilities score ranged from 0 to 1 and > 0.5 were considered a close interaction. L The sequences of FAP 3'-UTR-WT and FAP 3'-UTR-MUT used in luciferase reporter assay were presented. Luciferase reporter assay showing the luciferase activity of the FAP 3'-UTR luciferase reporter plasmid (WT or MUT) following transfection with miR-NC or miR-329-5p mimic into CAFs. M Western blotting was used to investigate the effect of miR-329-5p on FAP expression. N and O The effect of the circNOX4/miR-329-5p axis on the expression level of FAP. P The effect of the circNOX4/miR-329-5p axis on the fibroblast activation reflected by collagen gel contraction. *P < 0.05, **P < 0.01, ***P < 0.001