Figure 6. MiR-133b was a direct regulator of HOXB8 in CCA cells. (A) MiR-133b restrained HOXB8 mRNA expression confirmed by qRT-PCR. (B) MiR-133b refrained HOXB8 protein expression testified via western blot in QBC939 and CCLP-1 cells. (C) The expression of HOXB8 mRNA in CCA tissues and paired adjacent nontumor bile duct tissues. (D) The correlation between relative HOXB8 mRNA expression and relative miR-133b expression in CCA tissues. (E) The HOXB8 mRNA expression in QBC939, CCLP-1, RBE, TFK-1 and normal HIBEC. (F) The HOXB8 protein expression in CCA cells (QBC939, CCLP-1, RBE, TFK-1) and normal HIBEC. (G) Luciferase reporter plasmids were constructed with miR-133b-binding site region of HOXB8 sequence, including wild type and mutant type. (H) The luciferase activity of HOXB8 wild type was inhibited by miR-133b mimics cotransfection. (I) AGO2 RIP assays were conducted to further demonstrate the binding of miR-133b to 3’UTR of HOXB8. *P < 0.05, **P < 0.01, ***P < 0.001.