Figure 3

MiR-144 directly targets Zeb1.

(A) Zeb1, Zeb2, Rock1 and Rgs17 are targets of miR-144. Predicted duplex formation between Zeb1-3′-UTR, Zeb2-3′-UTR, Rock1-3′UTR, Rgs17-3′UTR and miR-144. (B) Luciferase assays in 293T and A549 cells transfected with a luciferase reporter controlled by the indicated elements. (C) Sequences of wild-type and mutant target sites for miR-144 in the Zeb1-3′ UTR. Two point mutations (underlined) predicted to abolish miRNA-mRNA binding were introduced into the miR-144 recognition region. (D, E) Luciferase reporter assays were performed in 293T (D) and A549 (E) cells co-transfected with the miR-control or miR-144 mimic (miR-144) together with the luciferase gene driven by the wild-type or mutant Zeb1-3′UTR sequences. The normalized luciferase activity in the control group was set as the relative luciferase activity. Each bar represents the mean ± S.D. for triplicate experiments. P values were determined by Student’s t-test. *p < 0.05; **p < 0.01. (F, G) The cells were transfected with the miR-control or miR-144 mimic (F) or anti-miR-144 (G) at a final concentration of 100 nM. The expression of the indicated proteins was determined by immunoblotting. (H) The expression of Zeb1 in tumour samples and their paired normal lung tissues from the 105 NSCLCs. (I) The correlation between Zeb1 and miR-144 expression was evaluated by Pearson correlation analysis. R and p-values are shown.