FIGURE 3.

Ets-1 serves as a novel direct target of miR-200b. A, in silico study revealing two possible binding sites in Ets-1 3′ UTR for miR-200b as predicted by Targetscan, Pictar, MiRanda, MiRBase Target Data base, and miRDB. B, miR target reporter luciferase assay after the miR-200b mimic delivery in HEK-293 cells. Open and solid bars represent control mimic and miR-200b mimic-delivered cells, respectively. Results were normalized with data obtained from an assay with Renilla luciferase and expressed as mean ± S.E. *** indicates p < 0.001 compared with control mimic-transfected cells; +++ represents p < 0.001 compared with control plasmid-transfected cells. C, representative diagram showing Ets-1 immunoreactivity after miR-200b mimic delivery from three independent experiments. Nuclear counterstain with DAPI and the corresponding merged image are shown in the lower panels. D, Western blot analysis of Ets-1 protein expression in miR-200b mimic-delivered (left) and -depleted (right) HMECs. β-Actin serves a loading control. Representative blot from three independent experiments is shown. Quantification of the band intensity relative to control. Results are mean ± S.E. *** indicates p < 0.001; * represents p < 0.05 compared with control.