Figure 6: Effect of miR-221 zipper to the target gene of miR-221.

(a) Sequence alignment showing the 3′-UTR of human p27 mRNA has two binding sites to miR-221/222. (b) Western blot analysis showing increased p27 expression by anti-miR-221 and anti-miR-222 in MDA-MB-231 cells. β-Actin served as protein loading control. See also Supplementary Fig.9. (c) Western blot analysis showing increased p27 expression in MDA-MB-231 cells by miR-221 zipper at 24 and 48 h after transfection. β-Actin served as protein loading control. See also Supplementary Fig.10. (d) Schematic representation of the luciferase reporter constructs carrying either p27 3′-UTR WT or p27 3′-UTR MU (point mutated in the two binding sites of miR-221/222) right downstream of the luciferase coding region. (e) Luciferase reporter assay showed increased luciferase activity by miR-221 zipper to WT p27 3′-UTR. The increased level of luciferase activity was not as high as that in mutated p27 3′-UTR cells mainly due to the unaffected expression of miR-222 in miR-221 zipper treated cells. The firefly luciferase activity was normalized to Renilla. Values are presented as the mean±s.e.m. (n=3). *P<0.05, **P<0.01 (miR zipper group versus control group, p27 3′-UTR mutation group versus p27 3′-UTR WT group, t-test, n=3).