Luciferase Assay for miR-222 Targeting

Dual luciferase reporter assays were performed to test for interactions between miR-222 and its target sequence, as previously described [44 (link)]. In brief, a luciferase reporter gene construct for ABCG2 (pGL-ABCG2) was created by cloning the 3′-UTR of ABCG2 [NM_004827, containing the miRNA-222 binding site (5′-UGUAGCA-3′)] into the Xho I and Not I sites of a pGL-3-Control firefly luciferase reporter vector (Promega, Wisconsin, USA). The corresponding mutant construct (pGL-ABCG2m) was created by replacing the seed regions of the miR-222 binding sites with 5′-ACATCGT-3′. These constructs were then verified by sequencing. Cells were transfected with the reporter constructs using Lipofectamine 2000 (Invitrogen, CA, USA). The pRL-TK vector (Promega, Wisconsin, USA) was co-transfected to serve as an internal control for normalization of the transfection efficiency. Luciferase activity was then determined using a GloMax 20/20 luminometer (Promega, Wisconsin, USA), as previously described [15 (link)].