Fig. 4

CircSLCO1B3 regulates miR-502-5p/HOXC8/SMAD3 axis to promote proliferation, migration and invasion in ICC. a Schematic illustration exhibited overlapping of the target mRNAs of miR-502-5p predicted by miRDB, miRTarBase and TargetScan databases. b qPCR was conducted to determine the relative expression levels of 12 potential target mRNAs after downregulation of circSLCO1B3 or miR-502-5p and upregulation of miR-502-5p in ICC cells. c Western blotting was performed to determine the expression of HOXC8 in ICC cells after overexpression or downregulation of circSLCO1B3. d FISH scores statistical analysis of circSLCO1B3 in ICC tissues and normal tissues. e The correlation of HOXC8 and circSLCO1B3 in FISH and IF scores. f, g qPCR showed that the expression of HOXC8 in ICC cell lines and tissues. h CCK-8 assays and trans-well assays were performed to determine the ability of proliferation and migration in ICC cells transfected with HOXC8 vectors and HOXC8 siRNA (magnification, × 200, scale bar, 100 μm). i Schematic illustration and the relative luciferase activities of HOXC8-WT and HOXC8-Mut luciferase reporter vectors. j RNA pull-down assay was executed in HuCCT1 cells to detect the enrichment of HOXC8 by the miR-502-5p probe. k Western blotting was performed to determine the expression of smad2, p-smad2, smad3, p-smad3 in HuCCT1 cells after depleting circSLCO1B3. l Luciferase plasmids with the first 2,200 nucleotides of SMAD3 promoter domain or with blank plasmids were formed to detect the associative relation of SMAD3 and HOXC8. m ChIP assays were executed to detect the relation of SMAD3 promoter domain and HOXC8. n qPCR was conducted to detect the expression relation of HOXC8 and SMAD3 in 13 pairs ICC tissues and normal tissues. Data were showed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001