Fig. 2.

Bmf is a target of miR-221. A, transfection experiments in Hep3B, SNU398, and SNU449 HCC-derived cell lines. miR-221 and anti-miR-221 were transfected based on the basal expression levels of the different cell lines. Controls refer to negative control from Ambion. Enforced miR-221 expression caused a decrease of Bmf protein levels in Hep3B and SNU398 cells, whereas enforced anti-miR-221 caused an increased Bmf expression in Hep3B and SNU449 cells. Data are expressed as a mean of three replicates and P values resulting from t test are reported. B, analysis of Bmf mRNA and protein levels in Hep3B and SNU449 cells treated with anti-miR-221 and negative control. Following anti-miR-221 transfection, both cell lines do not display any variation of Bmf mRNA levels, whereas Bmf protein levels increase. This findings confirm that miR-221 controls Bmf expression at the translational level. C, luciferase assay in Hep3B cells. pMIR-BMF 3′-UTR vector was cotransfected with miR-221, anti-miR-221, or negative control. Luciferase activity displayed a significant decrease following miR-221 enforced expression and a significant increase following anti-miR-221 enforced expression when compared with cells cotransfected with negative control.