Photinus pyralis renilla reniformis dual luciferase reporter assay system

A total of 5,000 293 cells were seeded in a 96-well plate at 70% confluence. The YAP1 wild type (WT) 3′-UTR firefly luciferase construct (pMir-YAP1 WT- 3′-UTR) was generated by inserting YAP1 WT 3′-UTR into pMir-Report vector (Ambion; Thermo Fisher Scientific, Inc.). Mutations were introduced in potential miR-590-5p binding sites using the QuikChange site-directed mutagenesis kit (Stratagene; Agilent Technologies, Inc., Santa Clara, CA, USA) according to the manufacturer's protocol. Then, a final concentration of 100 nM miR-590-5p NC or mimics were transfected into 293 cells along with 30 ng pMir-YAP1 WT or mutant 3′-UTR luciferase reporter and 10 ng Renilla luciferase reporter using Lipofectamine^® 2000, as previously stated. Cells were collected 48 h post-transfection, and luciferase assays were performed using a Photinus pyralis-Renilla reniformis dual luciferase reporter assay system (Promega Corporation, Madison, WI, USA) according to the manufacturer's protocol. The ratio of Photinus pyramid to Renilla of each lysate luciferase activity was determined by an Orion II Microplate Illuminometer (Titertek-Berthold, South San Francisco, CA, USA). Relative activities were expressed as the fold change in luciferase activity.

The putative binding sites of miR-590-5p for lncRNA-ATB were predicted using RNA hybrid (20 (link)) and miRDB (http://www.mirdb.org/). Two hundreds and ninety-three cells were seeded in a 96-well plate at 70% confluence. The lncRNA-ATB was cloned into pMir-Report (Ambion; Thermo Fisher Scientific, Inc.), yielding pMir-Report-lncRNA-ATB. Mutations were introduced in potential miR-590-5p binding sites using the QuikChange site-directed mutagenesis kit (Stratagene; Agilent Technologies, Inc., Santa Clara, CA, USA). miR-590-5p NC or mimics were transfected into 293 cells with 30 ng wild-type (WT) or mutant (Mut) 3′-untranslated region (UTR) of lncRNA-ATB using Lipofectamine^® 2000, as previously stated. Cells were collected 48 h post-transfection, and luciferase assays were performed using a Photinus pyralis-Renilla reniformis dual luciferase reporter assay system, according to the manufacturer's protocol (Promega Corporation, Madison, WI, USA). The luciferase activity of each lysate was normalized to Renilla luciferase activity.