Dual luiferase reporter assay system

For dual-luciferase reporter assays, the 3′-UTR of PDCD4 containing miR-208a-3p binding sites were cloned into a pmirGLO dual-luciferase vector (Promega Corporation, Madison, WI, U.S.A.) to generate wild-type (WT) pmirGLO-PDCD4 3′-UTR. The mutant (MUT) 3′-UTR of PDCD4 gene with miR-208a-3p target sites were generated using a Site-Directed Mutagenesis kit (Agilent Technologies, Inc., Santa Clara, CA, U.S.A.), and cloned into a pmirGLO dual-luciferase vector (Promega Corporation) to generate MUT pmirGLO-PDCD4 3′-UTR. The WT pmirGLO-PDCD4 3′-UTR and MUT pmirGLO-PDCD4 3′-UTR were co-transfected with miR-208a-3p mimics, inhibitor or negative control (NC) by using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Cells were collected 48 h after the transfection, and the luciferase activities were analyzed with the Dual-Luiferase Reporter Assay System (Promega, U.S.A.) according to the manufacturer’s instructions.

The 3’-UTR of the human EPLIN mRNA (NM_001114676.1) was amplified from cDNA derived from total RNA of HUVECs, and subcloned into pGL3-promoter vector (Promega, USA) to construct a pGL3-EPLIN-WT reporter plasmid. A mutant reporter (pGL3-EPLIN-mt) with a mutation in the 3’-UTR complementary to the seed sequence of miR-93-5p was generated by using a Site Directed Mutagenesis Kit (Clontech, USA) according to manufacture’s instructions. The PCR primers are listed in Supplementary Table 1. To perform the luciferase reporter assay, HeLa cells were cotransfected with 100 ng of pGL3-EPLIN-WT or pGL3-EPLIN-mt, together with miR-93-5p mimics (50, 100 nM) or control oligos, and pRL-TK (5 ng). Cells were collected 48 h after the transfection, and firefly and renilla luciferase activities were analyzed with the Dual-Luiferase Reporter Assay System (Promega, USA) according to the manufacturer’s instruction.