Figure 4.

MiR-206 suppressed CCA cell stem-like characteristics and TGF-beta1 secretion via LASP1/STAT3 signalling. (A) Representative images of sphere formed by miR-206-mimic/HUCCT1 and miR-206-mimic/RBE cells in serum-free conditioned medium. Colony diameters were analyzed. Scale bar=100 µm. (B) The population of CD44+CD133+ cells in the miR-206-mimic/HUCCT1 and miR-206-mimic/RBE cell cultures was detected by FCM. (C) mRNA expression of the stem cell regulators Nanog, Sox2, and Oct4 was assessed by qPCR. (D) PITA, PicTar, TargetScan and microT were used to predict the target gene of miR-206. (E) The binding site of miR-206 in the 3'UTR of LASP1 was predicted, and this binding site was confirmed by a dual-luciferase reporter assay. (F) The correlation between miR-206 expression and LASP1 expression in tissues was analyzed (P<0.05, R²=0.5495). (G) Expression of LASP1 in miR-206 overexpression or knockdown HUCCT1 cell line was evaluated by qPCR and WB. (H) The overexpression of LASP1 was confirmed by qPCR and WB assays. (I) The Nanog, Sox2 and Oct4 mRNA levels in miR-206-mimic/HUCCT1 cells overexpressing LASP1 were analyzed. (J) Expression of p-STAT3, STAT3, and Nanog in miR-206-overexpressing HUCCT1 cells after IL-6 treatment. (K) Representative images of spheres and expression of p-STAT3, STAT3, and Nanog in the HUCCT1 cell line overexpressing the miR-206 and LASP1. Diameter of sphere was analyzed. Scale bar=100 um. (L) Representative images of spheres and protein expression of Nanog in LASP1-overexpressing HUCCT1 cells treated with Stattic (20 μM for 24 hours). Diameter of sphere was analyzed. Scale bar=100 um. (M) Secretion of TGF-beta1 by LASP1-overexpressing HUCCT1 cells treated with Stattic (20 μM for 24 hours). The data are shown as the mean ± SD, * P <0.05, ** P <0.01, *** P <0.001.