3′-UTR luciferase reporter assays

The 3′-UTRs of CCND2 (forward primer, 5′-GGAGTTCTTGGGAATCTTG-3′ and reverse primer, 5′-CCCTTTAGTGGGAGGTAA-3′) and E2F3 (forward primer, 5′-GCCAGTTTACTCCAGGTA-3′ and reverse primer, 5′-AACAATCTAGCCAGGTGA-3′) were amplified by PCR as above-mentioned from a human SKOV3 cell cDNA library and cloned downstream of the Renilla luciferase coding sequence, between the Xho I and Not I sites, of the psiCHECK2 luciferase reporter vector (Promega Corporation, Madison, WI, USA). The miR-145 target site in the CCND2 and E2F3 3′UTRs was mutated by altering the 3-nt miR-145 seed match sequence using the QuikChange Site-Directed Mutagenesis kit (Stratagene; Agilent Technologies, Inc., Santa Clara, CA, USA), according to manufacturer's protocol. The aforementioned 293 cells were cotransfected with 100 ng psiCHECK2-CCND2 3′UTR or psiCHECK2-E2F3 3′UTR, or psiCHECK2-CCND2 3′UTR-Mutant (Mut) or psiCHECK2-E2F3 3′UTR-Mut luciferase plasmid, and the miR-145 mimics or miR-145-negative controls (NC) using Lipofectamine^® 2000. After 24 h, luciferase activity was measured using the Dual-Glo Luciferase Reporter Assay System (Promega Corporation) according to the manufacturer's instructions. Data were normalized for transfection efficiency by comparison with Renilla luciferase activity.