Figure 5.

Chk1 and Wee1 are the targets of p53-dependent miR-16 and miR-26a. (a) Effect of miR-16 overexpression alone or together with its antago-miR (inhibitor) on expression levels of Chk1 and cyclin E was assessed by western blotting using specific antibodies. Beta-tubulin was used as a loading control. (b) Effect of individual or simultaneous overexpression of miR-16 and miR-26a on expression levels of Wee1 and Chk1 in H1299 cells. Expression levels of Wee1 and Chk1 in the cells transfected with control oligo were arbitrarily set as 1. Intensities of bands corresponding to the Wee1 and Chk1 proteins were calculated using ImageQuant software. (c) To determine the effect of p53 activation on cellular levels of the Chk1 and Wee1 proteins isogenic HCT116 cell lines with different p53 status were treated with doxorubicin for 16 h followed by western blotting against the respective proteins. Note, that anti-p53 antibody gave a non-specific band in samples prepared from p53-deficient cells. (d) Shown are the regions of homology between Wee1 and Chk1 3-UTRs and miR-16 and miR-26a sequences. (e) A schematic presentation of putative miR-16- and miR-26a-binding sites in the 3-UTR regions of Chk1 and Wee1. Below are shown the results of luciferase assay in which the activities of 3-UTRs of Chk1 and Wee1 fused to the luciferase gene constructs were measured in the presence of individually or simultaneously expressed miR-16 and/or miR-26a. Binding of these miRs to 3-UTRs of the respective genes should result in repression of the protein synthesis and hence decrease luciferase activity. Fold repression was calculated against the value of luciferase activity of the cells transfected with respective vectors in the absence of miRs, which was set as 1