Luciferase-based Validation of miR-761 Targets

The luciferase assay was performed as in a previous study [32 (link)]. Fragments of ADIRF-AS1 harboring the predicted target site of miR-761 were amplified and cloned into the pmirGLO reporter plasmid (Promega Corporation, Madison, WI, USA), which is referred to as wild-type-ADIRF-AS1 (wt-ADIRF-AS1). The ADIRF-AS1 fragments carrying the mutant (mut) predicted target site of miR-761 were inserted into the pmirGLO reporter plasmid, which yielded mut-ADIRF-AS1. The wt-IRS1 and mut-IRS1 reporter plasmids were designed and constructed in a similar manner. OS cells were co-transfected with miR-761 mimic or NC mimic and wt or mut reporter plasmids using Lipofectamine® 2000. After 48 h, the luciferase activity was measured in accordance with the protocol of the dual-luciferase reporter analysis system (Promega Corporation).