Prl tk renilla luciferase 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.

Partial fragments of PPAR-γ 3′UTR containing predicted miR-128 binding sites were amplified by PCR and subcloned into pGL3-control vectors (Promega, Madison, WI, USA) to generate wild-type (WT) PPAR-γ reporter. Moreover, mutant type (MUT) PPAR-γ reporter with mutant miR-128 binding sites was produced using Quickchage Multi Site-Directed Mutagenesis kit (Stratagene, Lajolla, CA, USA). Next, constructed WT-PPAR-γ or MUT-PPAR-γ reporter was co-transfected into MCN and N2a cells together with control Renilla luciferase pRL-TK vectors (Promega) and miR-Control or miR-128. At 48 h after transfection, relative luciferase activity was determined via dualluciferase reporter assay (Promega).