Figure 4

ADAM12-L, but notADAM12-S, is a target for miR-200b/c. (A,B) SUM159PT, SUM1315MO2 and Hs578T cells were transfected with miR-200b mimic, miR-200c 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 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-200b, miR-200c 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 three independent experiments ± SEM. Significance was determined by one-sample t tests. ***P < 0.001. Lower Three nucleotides in each putative miRNA target site (shown in bold) were mutated to destroy the site. The mutated residues are shown in red above the wild-type sequences. The positions in the ADAM12-L 3′UTR relative to the stop codon are indicated. (D) Hs578T cells were transfected with miR-200b mimic, miR-200c mimic, or mimic control. Forty-eight hours after transfection, cells were treated with ³⁵S methionine/cysteine for the indicated periods of time, followed by immunoprecipitation with anti-ADAM12-L antibody or pre-immune serum, SDS-PAGE and autoradiography. The nascent, full-length form of ADAM12-L (~120 kDa) is shown.