Figure 5

miR-221/222 downregulates MYBL1 through direct interaction with the MYBL1 3′UTR. (A) Vector MCF-7 cells were transfected with 10, 20 or 40 nM (indicated by the triangles) miR-221 or miR-222, both mimics combined, or negative control miRNA as shown, lysed, and subjected to Western blotting for MYB family protein expression as indicated. The graph shows the means of the 40 nM miRNA data, with S.E.M. error bars, normalized to the β-actin loading control. Data is from 3 biological replicates. (B) Effect of miR-221 and miR-222 mimics on MYBL1 3′UTR activity. The wild-type 3′UTR of MYBL1 was cloned into the psiCHECK2 plasmid just downstream renilla luciferase, and co-transfected with 40 nM of the indicated miRNA mimics into vector control cells. 48 h after transfection, cells were lysed and renilla and firefly luciferase activity measured as described in Materials and Methods. Renilla luciferase activity was normalized to that of firefly luciferase and is shown as the log2 transformed change relative to empty psiCHECK2 vector. The graph shows mean values with S.E.M. error bars of 18 independent, parallel experiments. (C) Effect of deletion of putative miR-221/222 binding sites on MYBL1 3′UTR activity in p95HER2 cells. The psiCHECK2 vector containing wild-type MYBL1 or this construct with disruptive mutations introduced in each of the three putative miR-221/222 binding sites were transfected into p95HER2 cells. Renilla luciferase activity was normalized to Firefly luciferase activity and is shown as the log2 transformed change relative to empty psiCHECK2 vector. ***P < 0.001, significantly different from wild type 3′UTR. Data is from 6–18 biological replicates.