Pmir report plasmid

Potential regulators of NDRG3 were identified using the TargetScan (version 7.2; http://www.targetscan.org/vert_72/) and MicroRNA (August 2010 Release; http://www.microrna.org/microrna/home.do) tools according to the protocols of these web tools. A miR-140-5p recognition site in the 3′-untranslated region (UTR) of NDRG3 was identified The NDRG3 3′-UTR containing the putative miR-140-5p binding site was cloned into a pMIR-REPORT plasmid (Promega Corporation) to construct the reporter vector, pMIR-NDRG3-WT. The GeneArt™ Site-Directed Mutagenesis system (Thermo Fisher Scientific, Inc.) was used to the produce mutant-type NDRG3 reporter (NDRG3-Mut). The NDRG3-WT (50 nM) and Mut (50 nM) reporters were co-transfected with miR-140-5p mimics (100 nM) into the BGC-823 and AGS gastric cancer cells using a lipid-based method (Lipofectamine^® 2000; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. At 24 h post-infection, luciferase activities were determined using a dual-luciferase assay system (Promega Corporation) according to the manufacturer's protocol. The red firefly luciferase signal was used as a normalization control.

Bioinformatics prediction was carried out using miRDB and TargetScan7 tools. Then, LINC01410 or VOPP1 fragment containing wild-type or mutant miR-2467-binding site was inserted into the pMIR-Report plasmid (Promega, Madison, USA) to generate luciferase reporter for luciferase reporter assay. Luciferase activities were measured through the Dual-Luciferase Reporter Assay System (Promega) following the Kit’s instruction.

The full-length 3ʹ-UTR of human APE1 was amplified by PCR from genomic DNA of 9901 cells, and the potential miR-765 binding site in the 3ʹ-UTR of APE1 was mutated by the overlap extension PCR method. Both wild-type and mutant 3ʹ-UTRs were subcloned into the pMIR-Report plasmid (Promega, Wisconsin, USA) directly downstream of the renilla luciferase coding sequence. The authenticity and orientation of the inserts were confirmed by sequencing. Luciferase assays were performed as previously described riefly, 5×10³ cells per well were seeded in 96-well plates 24 h before transfection. Cells were co-transfected with miRNA mimics or NC plus wild-type or mutant APE1 3ʹ-UTR plasmids using LipofectamineTM 2000 (Invitrogen, CA, USA). 36 h post-transfection, cells were assayed for both firefly and renilla luciferase using Dual-GloTM Luciferase Assay System (Promega, Wisconsin, USA) according to the manufacturer’s instruction.