Fig. 6: miR-139-5p regulates HSCs activation depending on β-catenin/SOX9/TGF-β1.

A Schematic drawing of the putative site SOX9 binding with the human TGF-β1 promoter. ChIP assay of the LX-2 cells transfected with Ad-SOX9 were performed with anti-SOX9 or IgG antibody. Representative of three experiments. B The mRNA expression of TGF-β1 in activated LX-2 cells infected with Ad-SOX9 or Ad-Con. C The wild-type TGF-β1 promoter (TGF-β1-WT) or the mutant of TGF-β1 promoter (TGF-β1-MUT) luciferase reporter activity in activated LX-2 cells infected with Ad-SOX9 or Ad-Con was detected by dual-luciferase reporter assay. D Pearson’s correlation analysis was performed to analyze the correlation between SOX9 and TGF-β1 expression levels in patient fibrotic liver tissues, n = 30. E The protein levels of TGF-β1, α-SMA, Collagen-I, and TIMP-1 in LX-2-pre-miR-139-5p cells cells transfected with Ad-β-catenin and Ad-shSOX9 were examined by western blotting. Representative of three experiments. F The protein levels of TGF-β1, α-SMA, Collagen-I, and TIMP-1 in LX-2-pre-miR-139-5p cells infected with Ad-TGF-β1 were detected by western blotting. Representative of three experiments. G Immunofluorescence staining for α-SMA (green) in LX-2-pre-miR-139-5p cells transfected with Ad-TGF-β1 was observed by confocal laser microscopy. DAPI was used to stain the nuclei, blue. Original magnification×400, Representative of three experiments. H CCK8 assay was performed to detect the proliferation of LX-2-pre-miR-139-5p cells infected with Ad-TGF-β1. I The transwell assay of the LX-2-pre-miR-139-5p cells infected with Ad-TGF-β1 or Ad-Con was performed to analyze the migration capability, representative of three experiments. The number of cells was calculated from different fields. Graph represents mean ± SD. *P < 0.05, **P < 0.01.