Pmir report vector

Bioinformatics analysis using TargetScan (http://www.targetscan.org/) indicated that HAX-1 represents a target gene of miR-125b. To verify this, the wild-type of the putative miR-125b binding sites of HAX-1 3′-UTR were cloned into the downstream of firefly luciferase gene in the pMIR-REPORT™ miRNA Expression Reporter Vector (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. To mutate the seed region of the miR-125b-binding sites (CUCAGGG to CUCUAGG), the QuikChange Site-Directed Mutagenesis kit (Agilent Technologies, Inc., Santa Clara, CA, USA) was used based on the wild-type conducted pMIR-REPORT™ vector following the manufacturer's protocol. To perform the luciferase reporter assay, the MCF-7/R cells were incubated in 48-well plates overnight at 37°C. The cells were then co-transfected with the wild-type (or mutant-type) of pMIR-REPORT vectors, Renilla luciferase pRL-TK vectors (Promega Corporation, Madison, WI, USA), and the miR-125b mimics using Lipofectamine 2000. After 48 h of transfection, the cells were collected and lysed using a lysis buffer provided by Promega Corporation (cat no. E1910). Luciferase activity was then measured using a Dual Luciferase Reporter Assay system according to the manufacturer's protocol (cat no. E1910, Promega Corporation). The relative Firefly luciferase activity was normalized to Renilla luciferase activity.