Figure 5.

MCM7 and miR-106b are repressed by Nutlin-activated p53 in an E2F-dependent manner. (A, B) WI-38 were infected with a retrovirus encoding for either a small hairpin RNA targeting p53 (p53i) or a control shRNA (Con) and treated with 10 μM Nutlin-3 for 24 or 48 h. QRT–PCR (A) and western blotting (B) analyses demonstrated p53 stabilization by Nutlin, which resulted in activation of p21 and repression of E2F1 mRNA and protein levels. MCM7 and its resident miR-106b were repressed in a p53-dependent manner upon Nutin-3 treatment. (C, D) WI-38 cells were infected with E1A or an empty vector control (Con) and treated with 10 μM Nutlin-3 for 24 h. E1A elevated E2F transactivation activity, resulting in the induction of Cyclin E and E2F1 itself as well as of MCM7 and miR-106b. Nutlin treatment of the control cells repressed transcription of E2F1 and its targets. E1A abolished this repression, indicating that the repression of E2F1 by p53 is necessary for the p53-dependent downregulation of MCM7 and miR-106b. In (A, C), statistically significant difference in expression (t-test; P-value<0.01) between the non-treated samples and the Nutlin-treated samples (at both 24 and 48 h) is marked by asterisks. (E, F) WI-38 cells were infected with a retrovirus encoding for either a small hairpin RNA targeting E2F1 (E2F1i) or a control shRNA (Con) and treated with 10 μM Nutlin-3 for 48 h. QRT–PCR (E) and western blot (F) analyses demonstrated repression of E2F1 and its targets miRs-106b/93/25, as well as activation of p53 and p21 upon Nutlin treatment. E2F1 knockdown mimicked the effect of Nutlin treatment in repressing the miR-106b/93/25 polycistron. Nutlin treatment in the presence of E2F1 shRNA had little effect on the miRs, indicating that E2F1 inhibition mediates the repression of the miRs by Nutlin-activated p53. GAPDH protein levels serve as loading controls in (B, D, F). QRT–PCR data are represented as mean±s.d.