Luciferase reporter gene analysis system

Artificially synthesized circPRKCH wild-type and mutant-type (namely circPRKCH-WT/MUT) and HGF 3′-UTR wild-type and mutant-type (HGF 3′-UTR-WT/MUT) constructs containing the putative binding sites of miR-145 (GenePharma, Shanghai, China) were inserted into psiCHECK2 vectors (Promega, Madison, WI, USA). Lipofectamine 2000 (Invitrogen) was then used to co-transfect miR-145 mimics or NC mimics with wild-type or mutant constructs into 293T cells. Forty-eight hours after transfection, luciferase activity was detected using the luciferase reporter gene analysis system (Promega).

A binding site between miR-150-5p and NOTCH2 was searched by bioinformatics prediction, and wild sequence of the binding site between miR-150-5p and NOTCH2 or mutated sequence were synthesized and inserted into pGL3-Basic vectors (NOTCH2-wt and NOTCH2-mut). The vectors identified by sequencing were transfected with mimic NC or miR-150-5p mimic into microglial cells. The cells were transfected for 48 h and then lysed. A luciferase assay kit (K801-200, Biovision) and a luciferase reporter gene analysis system (Promega, Madison, WI, USA) were used to calculate luciferase activity, using Renilla luciferase as the internal control. The ratio of Firefly luciferase RLU to Renilla luciferase RLU represented the activation degree of the target reporter gene. Each experiment was repeated thrice.