Pgl3 report luciferase vector

A DNA fragment comprised ofa partial wild-type 3′UTR of ESR1 or a corresponding mutant 3′UTR of ESR1 was chemically synthesized and cloned into the pGL3-REPORT luciferase vector containing Renilla luciferase and firefly luciferase (Ambion; Thermo Fisher Scientific, Inc.) downstream of the luciferase gene (designated pGL3-ESR1-3′UTR-WT and pGL3-ESR1-3′UTR-MT, respectively). The nucleotide sequences of the plasmid constructs were confirmed by DNA sequencing. All steps of the luciferase reporter assay were performed in accordance with procedures previously described (16 (link),17 (link)). Briefly, 293 cells (5×10⁴) were seeded into 24-well plates and transfected with 0.2 µg of either pGL3-ESR1-3′UTR-WT or pGL3-ESR1-3′UTR-MT, with or without 50 nM miR-107m or miR-107m NC using Lipofectamine 2000 transfection reagent according to the manufacturer's protocol. After 48 h, we used the dual luciferase reporter assay system to assess the luciferase activity (Promega Corp., Madison, WI, USA) and the results were expressed as the relative luciferase activity (firefly LUC/Renilla LUC). Meanwhile, ERα was the predicted target of miR-107 using the miR target prediction algorithm TargetScan (http://targetscan.org/), Pictar and miRanda.

Potential targets of miR-32 were predicted by choosing the bioinformatics analysis software miRanda (www.microrna.org/microrna/home.do) and TargetScan (www.targetscan.org). The wild type (WT) and mutant type (Mut) 3′-UTR of EZH2 were cloned into pGL3-REPORT luciferase vector (Ambion; Thermo fisher Scientific, Inc., California). For the luciferase assay, the cells were seeded into a 24-well plate at a density of 1.5 × 10⁵ cells/well and cultured overnight at 37°C prior to cotransfected with a reporter plasmid, along with miR-32 mimics and WT or Mut 3′-UTR of EZH2 using Lipofectamine 2000 transfection reagent at 37°C. Following 48 hours of incubation, the cells were harvested, and then detected by using Dual-Luciferase Reporter Assay System (Promega).