Figure 4

Identification of FoxM1 as a target of miR-370. (A) Predicted site of miR-370 in the FoxM1 3’ UTR mRNA. (B) Luciferase activities were determined at 24 hours and were normalized by Renilla luciferase activity. K562 cells were plated in 24-well plates, and then transfected with 0.05 μg of a Renilla luciferase expression construct pRL-TK and 0.5 μg of the pGL3-FoxM1-wt-luc or pGL3-FoxM1-mut-luc firefly luciferase expression construct, along with either miR-370 precursor or control precursor. Luciferase assays were performed after 24 hours using the dual luciferase reporter assay system. The expression of firefly luciferase activity was normalized to that of renilla luciferase activity for each sample. A decrease in relative firefly luciferase activity in the presence of miR-370 indicates the presence of a miR-370 modulated target sequence in the 3’-UTR of FoxM1. Data represents mean ± s.e. from three separate experiments. *P < 0.05. (C) and (D) The expression of FoxM1 and downstream genes were regulated by miR-370 overexpression at both transcription level and translation level. Western blots were performed and sequentially probed with antibodies against FoxM1, c-Myc and p27^kip1, β-actin was a loading control. Efficient miR-370 overexpression was verified in Figure2 (A). (E) Increase in FoxM1 protein level after anti-miR-370 treatment. Efficient miR-370 depletion was verified in Figure2 (B)