Figure 5. ZEB1-AS1 functioned as a sponge for miR-133b in CCA cells. (A) Subcellular localization of ZEB1-AS1 was tested by subcellular fractionation assays. GAPDH and U6 were used as endogenous controls for cytoplasm and nucleus, respectively. (B) The expression levels of predicted miRNAs were detected after knocking down ZEB1-AS1 in QBC939 and CCLP-1 cells. (C) The expression of miR-133b in CCA tissues and paired adjacent nontumor bile duct tissues. (D) The correlation between relative ZEB1-AS1 expression and relative miR-133b expression in CCA tissues. (E) The miR-133b expression in CCA cells (QBC939, CCLP-1, RBE, TFK-1) and normal HIBEC. (F) Luciferase reporter plasmids were constructed with miR-133b-binding site region of ZEB1-AS1 sequence, including wild type and mutant type. (G) Luciferase reporter assays showed that cotransfected miR-133b mimics significantly inhibited luciferase activity of ZEB1-AS1 wild type. (H) AGO2 RIP assays further suggested the binding of miR-133b to ZEB1-AS1. *P < 0.05, **P < 0.01, ***P < 0.001.