Fig 2: SMAD4 served as a target of miR-486-5p. a The fragments of SMAD4 3'UTR-WT and SMAD4 3'UTR-MUT were presented. b Dual-luciferase reporter assay indicated that the luciferase activity of SMAD4 3'UTR-WT could be reduced by miR-486-5p (P < 0.0001). c RIP assay suggested that the enrichment of miR-486-5p and SMAD4 was increased in Ago2 (P < 0.0001). d Biotin-labeled RNA pull-down assay showed that the enrichment of SMAD4 could be pulled down by the bio-miR-486-5p probe (P < 0.001). e, f QRT-PCR and WB analysis suggested that the mRNA and protein levels of SMAD4 were promoted in posterior capsular tissues from PCO patients compared to normal humans. g Pearson correlation analysis revealed that miR-486-5p expression was negatively correlated with and SMAD4 expression in posterior capsular tissues of PCO patients. h, i QRT-PCR and WB analysis showed that the mRNA and protein levels of SMAD4 were increased in SRA01/04 cells treated with TGF-ß2 in a dose-dependent manner (P < 0.05, P < 0.01, P < 0.0001). j QRT-PCR analysis revealed that the expression of miR-486-5p was enhanced by miR-486-5p mimic (P < 0.0001) while reduced by miR-486-5p inhibitor (P < 0.001) in TGF-ß2-stimulated SRA01/04 cells. k WB analysis presented that the protein level of SMAD4 was decreased by miR-486-5p overexpression (P < 0.0001) and increased by measured by miR-486-5p inhibition (P < 0.0001). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig 3: Effects of miR-486-5p overexpression and SMAD4 overexpression on the progression of TGF-ß2-stimulated SRA01/04 cells. a SRA01/04 cells were transfected with SMAD4 overexpression vector or pcDNA, and then treated with 10 ng/mL TGF-ß2. WB analysis showed that the protein level of SMAD4 was promoted by SMAD4 overexpression vector (P < 0.0001) in TGF-ß2-treated SRA01/04 cells. b–f SRA01/04 cells were co-transfected with miR-486-5p mimic and SMAD4 overexpression vector or pcDNA, and then treated with 10 ng/mL TGF-ß2. MTT assay (b) and transwell assay (c, d) suggested that overexpressed SMAD4 reversed the inhibition effect of miR-486-5p on the proliferation (P < 0.0001), migration (P < 0.01, P < 0.001) and invasion (P < 0.0001) (100 µM) of TGF-ß2-stimulated SRA01/04 cells. e WB analysis revealed that the regulation of miR-486-5p on the protein levels of E-cadherin, Vimentin and a-SMA could be reversed by SMAD4 overexpression (P < 0.0001) in TGF-ß2-stimulated SRA01/04 cells. f WB analysis showed that miR-486-5p inhibitor reversed the decreasing effect of NEAT1 silencing on SMAD4 protein expression (P < 0.0001). **P < 0.01, ***P < 0.001, ****P < 0.0001