Dual-Luciferase Reporter Assay

To generate the pmirGLO-ADAM10-3'UTR reporter plasmid, the human ADAM10-3'UTR region (423 bp) encompassing the binding site of miR-891a-5pwas amplified using PCR primers (3'UTR-F: 5'-GCAGCTAGCACTAAACCCTCACAAG-3'; 3'UTR -R: 5'-TTGCGTCGACACAGAAGTACAGTGTA-3') and then cloned into the luciferase vector pmirGLO(Promega) using NheI and SalI restriction enzymes (New England Biolabs). T47D cells were transfected with different combinations including pmirGLO-ADAM10-3'UTR (WT) plus miR-NC; pmirGLO-ADAM10-3'UTR (WT) plus miR-891a-5p; pmirGLO-ADAM10-3'UTR (Mut) plus miR-NC; pmirGLO-ADAM10-3'UTR (Mut) plus miR-891a-5p for 48 hrs. Then the cells were harvested and lysed, and luciferase activity was detected by GloMax®- Multi Detection System (Promega) using the Dual- Luciferase Reporter Assay Kit (E1910, Promega). Luciferase activity was normalized to renilla luciferase activity.