Fig. 4

VIRMA promotes ICC proliferation, invasion, and metastasis in vitro. a qRT-PCR (up) and western blot (down) analysis of VIRMA expression in ICC cell lines and normal bile duct epithelial cell line HIBEpiC. The relative quantification was calculated using the 2^−ΔΔCt method and normalized based on GAPDH. b The growth of both RBE and HuCCT1 cells after knockdown or overexpression of VIRMA over 5 days was measured using CCK-8 assays. c The growth of cells over 14 days after knockdown or overexpression of VIRMA was measured using colony formation assays. d Quantitation of colony formation assays. e Transwell migration assay and matrigel invasion assay of RBE cells and HuCCT1 cells after knockdown or overexpression of VIRMA. Scale bars, 100 μm. f Quantitation of transwell assays. g Wound healing assay of the RBE and HuCCT1 cell lines after knockdown or overexpression of VIRMA. Images of wound repair were taken at 0, 24 h after wounding. h Quantitation of wound healing assays. The distance of wound closure was shown by area at 24 h. i, j The mRNA and protein levels of EMT markers, angiogenic marker VEGF and proliferation marker Cyclin D1 in ICC cells with VIRMA knockdown or overexpression were evaluated by i western blot and j qRT-PCR. siVIRMA-1 and siVIRMA-2 indicate VIRMA knockdown in ICC cells, while oeVIRMA indicates ICC cells transfected with an overexpressing plasmid. *P < 0.05, **P < 0.01, ***P < 0.001