Figure 2:Identification of Smad4 as a direct target of miR-25in NSCLC cells. A Schematic illustration of the predicted miR-205 binding site in the 3′-UTR region of the Smad4 gene. Predicted duplex formation between miR-205 and the wild-type/mutant of miR-205 binding site is indicated. B Molecular targeting of 3’-UTR region of Smad4 gene by miR-205. The luciferase activities in A549 cells transiently transfected with different combinations of plasmids containing the wild-type or mutant Smad4 3′-UTR fragment and miRNAs were analyzed. Scrambled sequence was used as miR-NC. Relative Renilla luciferase activity is obtained after normalizing to the firefly luciferase activity. C Impact of miR-205 on the mRNA expression of endogenous Smad4 in NSCLC A549 and SPC-A1 cell lines. Tumor cells were transiently transfected with miR-NC or miR-205 mimics, followed by real-time PCR analysis to evaluate expression of Smad4 mRNA. D Downregulation of endogenous Smad4 protein by miR-205. A549 cells and SPC-A1 cells were transiently transfected with miR-205 mimics or miR-NC, followed by immunoblotting of Smad4 and β-actin. *p<0.05; **p<0.001; ***p<0.001.