Pmirglo mirna target expression vector

The pmirGLO miRNA target expression vector (Promega, San Lius Obispo, CA, USA) was used to construct the recombinant plasmid pmirGLO-CREB1 containing the CREB1 mRNA 3′-UTR fragments as previously described [14 (link)]. For the luciferase reporter assay, cells were co-transfected with 30 nM of miRNA mimics or negative control and 30 ng pmirGLO-CREB1 (3′-UTR) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Forty-eight hours after transfection, the luciferase activity was measured using the dual luciferase assay system (Promega, Madison, WI, USA) according to the manufacturer's instructions. Each experiment was performed in triplicate.

The TINCR mRNA 3′-UTR fragment was amplified by PCR and inserted into the pmirGLO miRNA target expression vector (Promega, San Luis Obispo, CA, U.S.A.) to construct pmirGLO-TINCR (3′-UTR) plasmid. For the luciferase reporter assay, cells were cotransfected with miRNA mimics (50 nM) or negative control (50 nM), and pmirGLO-TINCR (3′-UTR) (50 nM) with Lipofectamine 3000 (Life Technologies, Carlsbad, CA, U.S.A.), according to the manufacturer’s instructions. After transfection for 48 h, the activities of Renilla luciferase and firefly luciferase were determined by the dual-luciferase reporter assay system (Promega). The luciferase activity was normalized to the firefly luciferase activity.

Dual-luciferase assays were performed as previously described [9] (link). The 3′-UTR fragments of CSF1 gene containing the miR-214 binding site was amplified by PCR from MKN45 cell RNA using the primers in Table S1, and inserted into the Xba1 site of pmirGLO miRNA target expression vector (Promega, San Lius Obispo, CA, USA). The resulting vector was named pmirGLO-CSF1. For the luciferase reporter assay, MKN45 and BGC823 cells were seeded in a 12-well plate the day before transfection. The cells were co-transfected with 30 nM of miR-214 precursor or miR-214 inhibitor, negative control and 30 ng pmirGLO-CSF1 using X-tremeGENE transfection reagent (Roche Applied Science). After 48 h transfection, luciferase activity was measured using the dual luciferase assay system (Promega) and normalized to Renilla luciferase activity. Each experiment was performed in triplicate.