FIG. 4.

Foxl2 is a direct target of miR-133a. (A) Predicted miR-133a binding sites in Foxl2 3′UTR. Specific locations of the binding sites were marked with red color, and Foxl2 3′UTR was marked with blue color. (B) Alignment between the predicted miR-133a target sites and miR-133a is shown. The conserved, 7-bp seed sequence for miR-133a:mRNA pairing is also indicated. (C) Diagram depicting the pMIR luciferase reporter constructs, containing a cytomegalovirus (CMV) promoter, which was utilized to verify the putative miR-133a binding sites. Luc, luciferase; poly A, poly(A) tail. (D) HEK293 cells were cotransfected with miR-133a, pLuc-FOXL2 3′UTR, along with a pRL-TK reporter plasmid. After 24 h, the luciferase activity was measured. Values are presented as relative luciferase activity after normalization to Renilla luciferase activity. The data are expressed as the mean value±S.E. (error bars) of the results obtained from three independent experiments. *Differences in luciferase activity between miR-133a and negative control transfected cells were significant, p<0.05. (E) Foxl2 expression levels in C2C12 cells after transfection with miR-133a were determined by western blot analysis. As compared with the NC miRNA, miR-133a expression reduced the levels of Foxl2 in C2C12 cells. GAPDH was used as an internal control. Foxl2, forkhead transcriptional factor 2; UTR, untranslated region.