Figure 4

Effect of Metformin on gene expression of mesenchymal, EMT, epithelial markers, AMPK and FOXO3 in iCCA primary cells and nuclear translocation of FOXO3 in iCCA primary cell cultures. (A) The gene expression of mesenchymal and EMT markers (Vimentin, SNAIL1, SNAIL2, TWIST1) and epithelial markers (E-Cadherin, Cytokeratin-19) was analysed by RT-qPCR in Large and Small duct-type iCCA primary cultures exposed to Metformin 10 µM for 48 and 96 h and normalized to the expression of GAPDH (housekeeping gene). In both iCCA subtypes, the gene expression of Vimentin was significantly decreased by Metformin only after 96 h while the expression of SNAIL1, SNAIL2 and TWIST1 decreased after 48 and 96 h. In contrast, the gene expression of the epithelial marker E-Cadherin significantly increased after 48 and 96 h in both Large and Small duct-type iCCA while Cytokeratin-19 increased only in Large duct-type iCCA after 96 h of exposure to Metformin. Data represent mean ± SD of N = 5 independent experiments. *p < 0.05. (B) The gene expression of AMPK and FOXO3, two signalling molecules involved in the mechanism of action of Metformin, was analysed by RT-qPCR and normalized to the expression of GAPDH (housekeeping gene). After 48 and 96 h, Metformin 10 µM significantly induced the gene expression of AMPK and FOXO3 in both Large and Small duct-type iCCA primary cultures. Data represent mean ± SD of N = 5 independent experiments. *p < 0.05. (C) IF analysis of FOXO3 in Large and Small duct-type iCCA primary cultures exposed for 96 h to Metformin 10 µM. Migration of FOXO3 from the cytoplasm to the nucleus is evident and occurred in 64 ± 10% of the cells. Nuclei were stained with DAPI. Magnification 20 × and 10 × ; representative images of N = 3 independent experiments.