Figure 4

MiR-223 binds to the 3’UTR of FBXW7 mRNA. (A) Sequence of the miR-223-binding site within the FBXW7 3’UTR (190–197 bp) predicted with TargetScan and miRBase. Mutation was generated on the FBXW7 3’UTR sequence in the complementary site for the seed region of miR-223. A FBXW7 3’UTR fragment containing wild-type or mutant miR-223-binding sequence was cloned downstream of the luciferase reporter gene in pGL3-luc vector; (B) Relative luciferase activity was analyzed after wild-type or mutant 3’UTR reporter plasmids were co-transfected with miR-224 mimic or inhibitor in 7901 cells. The histogram shows the mean ± S.D. of the normalized luciferase activity from three independent experiments; (C) qRT-PCR detection of FBXW7 mRNA expression in 7901 cells transfected with miR-223 mimic or inhibitor. GAPDH was used as an internal control; (D) Western blot detection of FBXW7 protein expression in cells transfected with miR-223 mimic or inhibitor. β-actin was used as an internal control. Data are expressed as the mean ± S.D. of three individual experiments. *p < 0.05.