Figure 2.

Luciferase reporter assay of putative miR-21 target genes and the effect of antisense (AS) to miR-21 on reporter activity. (A) Model of transient transfection assays in MCF-7 cells. MCF-7 cells were transiently transfected with pGL3-pro-luciferase and pRL-TK parental or pRL-TK containing putative miR-21 MREs from target genes (Supplementary Table 1) cloned in the 3′UTR as described in ‘Materials and methods’ section. Expected results are indicated without E₂ (A) and when cells are treated with E₂ (B). (C) MCF-7 cells were transfected as indicated and treated with EtOH or 10 nM E₂ for 24 h. Renilla luciferase was normalized by firefly luciferase to correct for transfection efficiency. Values are the average ± SEM of triplicate determinations. *Significantly different from EtOH control, P < 0.01. (D) MCF-7 cells were transfected with 2′-O-Me-antisense-miR-21 (ASmiR-21). Renilla luciferase reporter gene expression from the indicated gene MREs was determined and data analyzed as described in ‘Materials and Methods’ section. The control was a random-sequence 2′-O-Me modified RNA (control AS) as described in Materials and methods section. Values are the average ± SEM of triplicate determinations. *Significantly different from control AS, P < 0.05. (E) MCF-7 cells were transfected with the pRL-tk-MREs or FL 3′-UTRs as indicated. Indicated cells were co-transfected with ASmiR-21 or a control AS. Cells were treated with EtOH or 10 nM E₂ as indicated for 24 h. Dual luciferase reporter assays were performed and data quantitated as described in ‘Materials and Methods’ section. Values are the average ± SEM of triplicate determinations normalized to EtOH for each construct except that cells transfected with the ASmiR-21 were normalized against the control AS-EtOH value. ^aSignificantly different from EtOH control, P < 0.01. ^bSignificantly different from control AS transfected values, P < 0.01.