Pmirglo dual luciferase mirna reporter vectors

Target Scan 7.2 and miRnalyze online software (version 1) were used to analyze the putative target genes of miR-107. The 3′UTR of Wnt3a containing the miR-107 binding site was cloned into pmirGLO Dual-Luciferase miRNA Reporter Vectors (Promega, Madison, WI, USA). A mutated 3′ UTR of Wnt3a was introduced into the potential miR-107 binding site. The reporter vectors containing the wild-type or mutant Wnt3a 3′ UTR were transfected into NPCs using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). Briefly, cells were seeded in six-well tissue culture plates at 2 × 10⁵ cells/per well in 2.5 mL Opti-MEM (Invitrogen) at 12 h before transfection. On the day of transfection, the cells were exposed to the reporter vectors/Lipofectamine 3000 mixtures containing 2.5 μg of the luciferase reporter plasmid DNA mixture. At 8 h after transfection, the transfection medium was changed to (DMEM)/F-12 culture medium with 5% FBS and the cells were exposed to HBO. After 48 h, the transfected cells were washed with PBS and harvested using a passive lysis buffer (Promega). Cell lysates (20 μL) were evaluated for luciferase activity using a Dual-Luciferase Reporter Assay Kit (Promega).

Target Scan 7.2 (http://www.targetscan.org) online software was used to analyze the putative target genes of miR-573. The 3′ UTR of Bax containing the miR-573 binding site was cloned into pmirGLO dual-luciferase miRNA reporter vectors (Promega, USA). A mutated 3′ UTR of Bax was introduced into the potential miR-573 binding site. The reporter vectors containing the wild-type or mutant Bax 3′ UTR were transfected into NP cells using Lipofectamine 3000 (Invitrogen, USA). After incubation with or without HBO, transfected cells were lysed. Firefly and Renilla luciferase activities were detected using the dual-luciferase assay system (Promega, USA) in accordance with the manufacturer’s instructions.