Fig. 1

Transcription factor STAT3 regulates NAV2 expression to promote FLS proliferation, migration, and invasion in RA. a Immunofluorescence double staining analysis for the expression of NAV2 and Vimentin. DAPI for stain nuclei (blue). Vimentin and NAV2 were stained with green and red, respectively. b Expression of NAV2 level in FLS were examined. c Western blotting for NAV2, p-SSH1L, SSH1L, p-Cofilin-1, and Cofilin-1 in rats synovium tissue. Data were presented as means ± SEM from at least three independent experiments. n = 6–8 rats per group. Scale bars indicate 50 μm for immunohistochemistry. **P < 0.01, ***P < 0.001 vs Control group. Human RA FLS were treated with TNF-α (20 ng/ml) for 0, 1, 3, 6, 12, 24, and 48 h, the analysis of the inflammatory mediators was described in Materials and methods, respectively. d The expression level of NAV2 in RA FLS, at the different times following TNF-α treatment, was determined by Western blot and qRT-PCR. Data were presented as mean ± SEM of more than three independent experiments. **P < 0.01, ***P < 0.001 vs unstimulated cells. FLS were transfected with si Scr or si NAV2 prior to treatment with TNF-α (20 ng/ml), the level of protein expression was determined by Western blotting after 30 min or 12 h stimulation. e Silencing NAV2 decreased phosphorylation of SSH1L and increased phosphorylation of Cofilin-1 in contrast to control cells, GAPDH serves as the internal control. f Cells were subjected to immunofluorescence staining for NAV2 and PHALLOIDIN. DAPI for stain nuclei (blue). NAV2 and PHALLOIDIN were stained by green and red, respectively. Scale bars, 50 μm. g, h EdU incorporation with the quantification of percent EdU⁺ cells in NAV2 knockdown (siRNA) and control (si Scr) FLS before and after TNF-α treatment. i BrdU absorbance analysis. g, j Scratch wound migration assay showed that FLS had an augmented ability to migrate when compared to the control cells. g, k, l Transwell results showed that silencing NAV2 expression could obviously impede the invasion and motility of the cells. All images shown are representative ones from at least three replicates, results shown as mean ± SEM, ***P < 0.001 (n ≥ 3). m Effect of STAT3 on NAV2 gene promoter activity. A luciferase assay was carried out to analyze NAV2-dependent transcriptional activity by using 293 T cells. Data were presented as mean ± S.D, n = 3, ***P < 0.001 vs OE vector+mock group, ^###P < 0.001 vs OE vector+TNF-α group. n, o Schematic diagram of two pairs of primers designed for ChIP spanning the NAV2 promoter, STAT3 bound to the NAV2 promoter indicated by ChIP in RA FLS. IgG from rabbits served as a control. Data were presented as means ± SEM of three independent experiments. **P < 0.01, ***P < 0.001. p Working model for STAT3-NAV2 axis accelerates inflammatory response and related phenotypes. Unstimulated RA FLS has a lower level of NAV2 protein. NAV2 protein level increased after TNF-α-induced, leading to inflammatory protein response and inflammation-associated phenotypes. NAV2 was upregulated by transcription factor STAT3 and then activated the SSH1L/Cofilin-1 signaling pathway, subsequently promoting the progress of RA