For miR-623 target validation, the wild-type (WT) putative binding site of miR-623 in the 3′UTR of MMP1 predicted by TargetScan (edition7.2) (position 420–426 of MMP1 3’UTR), and the mutant (MUT) 3ʹUTR of MMP1 with the seed region deleted, were cloned into pmirGLO Dual-Luciferase miRNA Target Expression Vector (pmirGLO-empty, Promega, USA) to create Luciferase reporter constructs.13 (link),14 (link) The following primers were used to clone 3′-UTR-MMP1 in pmirGLO vector:
miR623_MMPI_WT_F: 5ʹCTAGTGTGCAGTCACTGGTGTCACCCTGGATAGGCAAGGGATAACTCTTCTAACACAAAATAAGTGTTTTA3’;
miR623_MMPI_WT_R: 5ʹCTAGTAAAACACTTATTTTGTGTTAGAAGAGTTATCCCTTGCCTATCCAGGGTGACACCAGTGACTGCACA3’;
miR623_MMPI_Mut_F: 5ʹCTAGTGTGCAGTCACTGGTGTCACCCTGGATAGGTAACTCTTCTAACACAAAATAAGTGTTTTA3’;
miR623_MMPI_Mut_R: 5ʹCTAGTAAAACACTTATTTTGTGTTAGAAGAGTTACCTATCCAGGGTGACACCAGTGACTGCACA3’;
(100 ng) PmirGLO-MUT, (100 ng) pmirGLO-WT, (20 nM) miR-623 mimic, or negative scrambled control were co-transfected into MDA-MB-231 cells (ATCC) using the Lipofectamine 3000 reagent protocol. The Firefly and Renilla luciferase activities were assessed using the Dual-Luciferase® Re-porter Assay System analysis (cat# E2940, Promega, USA) after 24 hours of transfection according to the manufacturer’s recommendations, and the Firefly activities were subsequently normalized with Renilla luciferase.
miR623_MMPI_WT_F: 5ʹCTAGTGTGCAGTCACTGGTGTCACCCTGGATAGGCAAGGGATAACTCTTCTAACACAAAATAAGTGTTTTA3’;
miR623_MMPI_WT_R: 5ʹCTAGTAAAACACTTATTTTGTGTTAGAAGAGTTATCCCTTGCCTATCCAGGGTGACACCAGTGACTGCACA3’;
miR623_MMPI_Mut_F: 5ʹCTAGTGTGCAGTCACTGGTGTCACCCTGGATAGGTAACTCTTCTAACACAAAATAAGTGTTTTA3’;
miR623_MMPI_Mut_R: 5ʹCTAGTAAAACACTTATTTTGTGTTAGAAGAGTTACCTATCCAGGGTGACACCAGTGACTGCACA3’;
(100 ng) PmirGLO-MUT, (100 ng) pmirGLO-WT, (20 nM) miR-623 mimic, or negative scrambled control were co-transfected into MDA-MB-231 cells (ATCC) using the Lipofectamine 3000 reagent protocol. The Firefly and Renilla luciferase activities were assessed using the Dual-Luciferase® Re-porter Assay System analysis (cat# E2940, Promega, USA) after 24 hours of transfection according to the manufacturer’s recommendations, and the Firefly activities were subsequently normalized with Renilla luciferase.
