Luciferase activity assay

The biological prediction website microRNA.org was used to analyze target genes of miR-132. We verified whether NRF2 was a direct target gene of miR-132 using a dual-luciferase reporter gene assay. Artificially synthesized NRF2 3′ untranslated region (3′UTR) gene fragments were introduced into the reporter plasmid pMIR-reporter (Beijing Huayueyang Biotechnology Co., Ltd., Beijing, China) using the endonuclease sites SpeI and Hind III. The complementary sequence mutation sites of the seed sequence were designed based on the wild-type (WT) NRF2. T4 DNA ligase was used to insert the target fragments into the pMIR-reporter plasmid after digestion with restriction enzymes. The correctly sequenced luciferase reporter plasmids NRF2 WT and NRF2-mutant (MUT) were cotransfected into T24 cells with miR-132 mimic. In addition, oe-KLF8, oe-NEDD4 or empty plasmids were cotransfected with the KLF8-mediated CyclinD1 promoter sequence into T24 cells. Cells were harvested 48 h later to extract the proteins. The luciferase detection kit (K801-200, Biovision) of GloMax20/20 Luminometer (Promega, Madison, WI, USA) was employed to assess luciferase activity.