miR-431-5p regulates cell proliferation and apoptosis in fibroblast-like synoviocytes in rheumatoid arthritis by targeting XIAP

Fig. 1. miR-431-5p was downregulated in RA. a Relative expression of miR-431-5p in synovial tissues of patients with RA (n = 8) and healthy controls (n = 6) by qRT-PCR. b Relative expression of miR-431-5p in HFLS and HFLS-RA cells by qRT-PCR. c Relative expression of miR-431-5p in HFLS-RA cells with or without TNF-α treatment by qRT-PCR. Each experiment was performed independently in triplicates. ***P < 0.001, ****P < 0.0001, compared with the NC groups

Fig. 2. Overexpression of miR-431-5p suppressed cell proliferation in RA FLSs. a qRT-PCR for the relative expression of miR-431-5p in HFLS-RA cells transfected with miR-431-5p mimics or inhibitor. b Cell proliferation pattern of HFLS-RA cells transfected with miR-431-5p mimics or inhibitor for 0 h, 24 h, 48 h, 72 h, and 96 h using the CCK-8 assay. c, d Cell proliferation of HFLS-RA cells transfected with miR-431-5p mimics or inhibitor for 48 h by EdU staining assay. Each experiment was performed independently in triplicates. *P < 0.05, ***P < 0.001, ****P < 0.0001, compared with the NC groups

Fig. 3. Overexpression of miR-431-5p induced apoptosis and suppressed G0/G1-to-S phase transition in RA FLSs. a Flow cytometry of Annexin V-FITC and PI-stained HFLS-RA cells transfected with miR-431-5p mimics or inhibitor for 48 h to determine cellular apoptosis. b–d Quantification of early (b), late (c), and total (d) apoptotic ratios of HFLS-RA cells transfected with miR-431-5p mimics or inhibitor. e, f Flow cytometry and quantification for cell cycle progression using PI-stained HFLS-RA cells transfected with miR-431-5p mimics or inhibitor for 48 h. Each experiment was performed independently in triplicates. *P < 0.05, ***P < 0.001, ****P < 0.0001, compared with the NC groups

Fig. 4. miR-431-5p directly bound XIAP in RA FLSs. a Binding between miR-431-5p and XIAP predicted by TargetScan. b Dual-luciferase reporter assay showing binding between miR-431-5p and the 3′ UTR of XIAP. c qRT-PCR for the relative expression of XIAP in HFLS-RA cells transfected with miR-431-5p mimics or inhibitor. d, e Western blotting to determine the levels of XIAP in HFLS-RA cells transfected with miR-431-5p mimics or inhibitor. f qRT-PCR for the relative expression of XIAP in synovial tissues of patients with RA (n = 8) and healthy controls (n = 6). g, h Western blots showing the levels of XIAP in synovial tissues of patients with RA (n = 3) and healthy controls (n = 3). i qRT-PCR for the relative expression of XIAP in HFLS and HFLS-RA cells. j, k Western blots showing the levels of XIAP in HFLS and HFLS-RA cells. Each experiment was performed independently in triplicates. *P < 0.05, **P < 0.01, ***P < 0.001, compared with the NC groups

Fig. 5. miR-431-5p regulated cell proliferation via XIAP in RA FLSs. a qRT-PCR for the relative expression of XIAP in HFLS-RA cells transfected with three siRNAs targeting XIAP. b, c Western blotting to determine the levels of XIAP in HFLS-RA cells transfected with three siRNAs against XIAP. d Proliferation of HFLS-RA cells transfected with miR-431-5p inhibitor and/or siRNAs against XIAP for 0 h, 24 h, 48 h, 72 h, and 96 h using CCK-8 assay. Asterisk for siRNA#1 and pound sign for siRNA#2. e, f Proliferation of HFLS-RA cells transfected with miR-431-5p inhibitor and/or siRNAs against XIAP for 48 h using EdU staining assay. Each experiment was performed independently in triplicates. *P < 0.05, ****P < 0.0001, compared with the NC groups

Fig. 6. miR-431-5p regulated apoptosis and cell cycle progression via XIAP in RA FLSs. a, b Flow cytometry and quantification for total apoptosis using Annexin V-FITC and PI-stained HFLS-RA cells transfected with miR-431-5p inhibitor and/or siRNAs against XIAP for 48 h. c, d Flow cytometry and quantification of cell cycle progression using PI-stained HFLS-RA cells transfected with miR-431-5p inhibitor and/or siRNAs against XIAP for 48 h. Each experiment was performed independently in triplicates. **P < 0.01, ***P < 0.001, ****P < 0.0001, compared with the NC groups