Identification of miR-130a binding sites in the Smad4 mRNA. (A) Schematic drawing showing the cloning of the predicted miR-130a binding sites in the 3′-UTR of pMIR-REPORT. (B-C) 32Dcl3 cells were transfected individually with the firefly expression vector pMIR-REPORT containing 1 of the 4 different constructs (wt1/wt2, mut1/wt2, wt1/mut2, and mut1/mut2) along with a renilla luciferase vector (for normalization) and pre–miR-130a/scrambled miRNA (B) or LNA-130a/LNA-neg (C), respectively. Relative firefly luciferase activity is shown after normalizing to the renilla luminescence. Data are shown as the mean ± SD from triplicate measurements. The expression from the transfections with the controls (miR-neg and LNA-neg) was assigned the value 1, and the relative expression measured from the constructs cotransfected with miR-130a or LNA-130a was recalculated accordingly.