To generate a reporter vector bearing miRNA-binding site, the 3-untranslated region (3′-UTR) of FoxO4 was synthesized by Sangon (Shanghai, China). The construct was inserted into multiple cloning sites downstream of the luciferase gene (SacI and HindIII sites) in the pMIR-REPORT luciferase miRNA expression reporter vector (Ambion, USA). To test the binding specifcity, the sequences that interacted with the seed sequence of miR-216b-5p were mutated (from AGAGAUU to UAUAUAA for the miR-216b-5p binding site), and the mutant FoxO4 3'-UTR was inserted into an equivalent luciferase reporter plasmid. For the luciferase assay, 0.1 μg of luciferase reporters containing 3′-UTR were co-mingled with miR-216b-5p mimic or miR-216b-5p inhibitor or miR-NC into HEK-293 cells using lipofectamine 2000 (Invitrogen, USA). As an internal control, a 10 ng of renilla luciferase reporters was also included. After 48 h transfection, the cells were collected and double luciferase activity was measured by luminometer according to the manufacturer's instructions (Promega Corporation).

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