Validating miR-124-3p Targeting of EZH2

EZH2-MUT (mutant-type) and EZH2-WT (wild-type) reporter plasmids were created by synthesizing and propagating the anticipated and mutated miR-124-3p target-binding sequences in EZH2 into a luciferase reporter. PCR was used to amplify the wild-type human EZH2 3’-UTR segment from PC3 cells, which contained the anticipated miR-124-3p target locations. The mutant EZH2 3’-UTR sequence was generated by overlap-extension PCR. Next, wild-type and mutant 3’-UTRs were sub-replicated into the psiCHECK-2 luciferase vector. Into 24-well culture plates, PC3 cells were seeded and co-transfected with miR-124-3p or an NC repressor using the Lipofectamine® 2000 system in the luciferase enzyme reporter studies. We extracted the cell transfects after 2 days. The luciferase enzyme activity evaluation was conducted utilizing Dual-Luciferase enzyme reporter assay platform.