Dual luciferase reporter system kit

Using the hypoxia response elements (HREs) in the NR2F1-AS1 promoter region, we constructed a wild-type (WT) and mutated (MUT) sequence containing two putative HIF-1α binding sequences into pGL3-based vectors (RiboBio). Luciferase reporter plasmid pGL3-based vectors expressing NR2F1-AS1-WT or MUT were co-transfected into MIA PaCa-2 and PANC-1 cells in 96-well plates with siNC or siHIF-1α. After 24 h, cells were cultured under normoxia or hypoxia for an additional 24 h. Cell lysates were harvested, and firefly and Renilla luciferase activities were measured using the dual-luciferase reporter system kit (Beyotime, Shanghai, China) on a microplate reader according to the manufacturer’s suggestions and protocols. The Renilla luciferase internal control was used to normalize luciferase activity.

The wild-type (wt) or mut-type (mut) sequences of FEZF1-AS1 and PAX6 3′-UTR were obtained and cloned into pmirGLO vectors to generate the luciferase vectors, named by FEZF1-AS1-wt, FEZF1-AS1-mut, PAX6-wt, and PAX6-mut. Then, the WERI-RB1 was co-transfected with luciferase vectors and miR-363-3p inhibitor or negative control inhibitor (NC inhibitor) for 48 h. Y79 was co-transfected with luciferase vectors and miR-363-3p mimic or NC mimic for 48 h. A fluorescence microplate reader measured luciferase activity through Dual-Luciferase Reporter System Kit (Beyotime, Shanghai, China).