FIG. 3.

Regulation of the ZEB2 3′UTR by miR-192/215. A: Alignment of the miR-192/215 sequences and the targeted area of the 3′UTR of ZEB2 (http://www.targetscan.org). The seed sequence at the 5′ end of the miRNA-192/215 and the targeted region in the ZEB2 3′UTR at nucleotides 955–961 downstream of the stop codon are shown. B: NRK52E cells were transfected with ZEB2 3′UTR luciferase reporter plasmid (1 μg), β-galactosidase plasmid (0.2 μg), and either miR-control (miR-C), miR-192, or miR-215 (100 nmol/l). TGF-β (10 ng/ml) was added 4 h after transfection, and cells were analyzed for β-galactosidase and luciferase (Luc) activity 3 days later. TGF-β significantly increased luciferase activity (P < 0.05 compared with control). MiR-192/215 significantly reduced luciferase activity from the ZEB2 3′UTR in the absence (*P < 0.05) or presence (#P < 0.001) of TGF-β, compared with control. C: Anti-miR-192/215 (anti-miRs) had no effect on the luciferase activity of the ZEB2 3′UTR construct compared with control. D: Western analysis and quantitation for ZEB2 expression in NRK52E cells transfected as in B with miR-control and miR-192/215. miR-192/215 significantly reduced ZEB2 protein expression (*P < 0.05, compared with control). E: Western analyses and quantitation of ZEB2 expression in NRK52E cells transfected either with miR-conrtol or miR-192/215 in the presence or absence of TGFβ. miR-control had no effect on the increased expression of ZEB2 induced by TGF-β; however, miR-192/215 prevented this induction (*P < 0.05, compared with control). All error bars represent ± SEM.