miR-454 directly targeted Smad4 in LX-2 cells. (A) Sequence alignment of putative miR-454-binding site in the 3′-UTR of Smad4 and the sequence of the mutant 3′-UTR of Smad4 were shown. (B) The expression of Smad4 protein was reduced by miR-454 in TGF-β1-treated LX-2 cells,as determined by Western blot. (C) Over-expression of miR-454 could induce the decrease of the luciferase activity of the wild-type Luc-Smad4 reporter, but not that of the mutant-type Luc-Smad4 reporter. Meanwhile, miR-454 inhibitor could increase luciferase activity of the wild-type 3′-UTR of Smad4. * P <0.05 vs one’s own control group (cells transfected with NS-miRNA and wild or mutant plasmids).