Figure 4

MiR-320a and miR-140 target the 3′UTR of TfR and physically bind to TfR mRNA in the RISC. (A) Schematic layout of TfR mRNA and presumptive targets of miR-320a and miR-140 via the sequential complementary nucleotides as indicated. The targets and tetranucleotide mutant targets segments were cloned into a pGL3-control reporter vector. The corresponding mut miR-320 and mut miR-140 were synthesized. (B) Luciferase activity of lysates of F81 cells after 36 h co-transfection with pGL3-TfR 3′UTR, pRL-TK and miR-320 or miR-140 mimics. NC mimics were used as controls. (C) Luciferase activity of lysates of F81 cells after 36 h co-transfection with mut320-pGL3-TfR 3′UTR, pRL-TK and miR-320a or mut miR-320a mimics. NC mimics were used as controls. (D) Luciferase activity of lysates of F81 cells after 36 h co-transfection with mut140-pGL3-TfR 3′UTR, pRL-TK and miR-140 or mut miR-140 mimics. NC mimics were used as controls. (E) qPCR analysis of TfR mRNA among RNAs extracted from Ago2 immunoprecipitates or total samples of F81 cells, normalized to β-actin. Data are from 3 independent experiments (mean ± SD). Statistical significance was analyzed by Student’s t test; *P <0.05; **P <0.01; ***P <0.001; ns, not significant.