Figure 3

ADAM12-Lis a poor target for miR-30b/d. (A,B) SUM159PT and SUM1315MO2 cells were transfected with miR-30b mimic, miR-30d mimic, or mimic control. (A)ADAM12-L and ADAM12-S mRNA levels were measured by qRT-PCR and normalized to β-ACTIN. Fold changes in miRNA-transfected cells versus control cells were calculated. Graphs represent average values obtained in three (for SUM159PT) or two (for SUM1315MO2) independent experiments ± SEM. Statistical significance was determined by one-sample t tests. *P < 0.05. (B) Cell lysates were enriched for glycoproteins and analyzed by Western blotting using an anti-ADAM12-L antibody. The nascent, full-length form and the mature, processed form are indicated. A Ponceau S-stained band in the glycoprotein-enriched fraction and tubulin in total cell lysates were used as loading controls. (C)Upper SUM159PT cells were transfected with miR-30b, miR-30d mimics, or mimic control and then with the indicated ADAM12-L 3′UTR reporter or an empty vector and a Renilla luciferase control vector. The firefly luciferase activity was measured after 48 h and was normalized to Renilla luciferase activity and to the empty vector. Graph shows the average values for at least two independent experiments ± SEM. Significance was determined by one-sample t tests. ***P < 0.001. Lower Three nucleotides in the putative miRNA target site (shown in bold) were mutated to destroy the site. The mutated residues are shown in red above the wild-type sequence. The position in the ADAM12-L 3′UTR relative to the stop codon is indicated.