Figure 3
Two functional STAT3 binding sites are present on miPPR21. (A) Putative STAT3 binding sites from −47 to −38 positions and from −16 to −7 positions upstream of pri-miR-21 transcription start site (TSS) (+1) on miPPR-21. (B) Promoter reporter assay of a Firefly luciferase vector driven by bovine miPPR-21 (pGL3miR21-Luc) containing the two STAT3 binding sites shown in (A). Constructs containing mutations in both binding sites (I), a single binding site (II, III), or wild-type (IV) were co-transfected into MCF7 cells with a Renilla luciferase construct for normalization. The pGL3-Luc base construct containing no promoter element (V) was also transfected as a control. Grey rectangles indicate STAT3 binding sites, and X indicates a mutation in that binding site. MCF7 cells were treated with 15 ng/mL recombinant human LIF, and luciferase activity in untreated and LIF-treated cells is shown relative to baseline activity of wild-type construct (IV) in untreated conditions. Firefly luciferase light units normalized to Renilla luciferase light units are shown. Asterisks indicate significant differences between untreated and LIF treated cells transfected with the same construct. Constructs sharing same letters did not show significant differences in luciferase activity under the influence of LIF.
