Validating miR-96 Targeting of Smad7

TargetScan Human 7.2 (www.targetscan.org/vert_72) was used to predict Smad7 as a target of miR-96. The wild-type (wt) or mutant (mut) Smad7 3′-untranslated region (UTR) was cloned into the pGL3 vector (Promega Corporation, Madison, WI, USA). Subsequently, the pGL3-Smad7-3′UTR-wt or pGL3-Smad7-3′UTR-mut vector, along with the miR-96 mimic or the mimic-control (as described above), were transfected with Lipofectamine^® 2000 into 293T cells (ATCC, Rockville, MD, USA). Following 48 h, the luciferase activity was measured with a Luciferase Reporter Gene Assay kit (Promega Corporation), using a GloMax-Multi Jr Single Tube Multimode Reader (Promega Corporation). Renilla luciferase activity was used for normalization of the firefly luciferase activity (18 (link)).