Validating NOTCH3-miR-7-5p Interaction

The 3'-UTR sequence of NOTCH3 containing the miR-7-5p binding site was cloned and constructed into a pGL3 vector (E1910, Promega, USA) to generate a wild-type NOTCH3 reporter gene (NOTCH3-Wt). GeneArt™ site-directed mutagenesis system (Thermo Fisher Scientific) was used to generate the mutant NOTCH3 reporter gene (NOTCH3-Mut). NOTCH3-Wt or NOTCH3-Mut and miR-NC and miR-7-5p mimic enter EC cells [15 (link)]. 48 hours after transfection, 40 μL stop reagent Renilla luciferase was added after the determination of the firefly luciferase fluorescence value, and the luciferase activity was measured by the luciferase reporter gene assay system (Promega, Madison WI, USA).