Dual-luciferase reporter gene assay

The target sites for binding of miR-10b-5p and PDCD4-AS1, miR-10b-5p and IQGAP2 were predicted by online software TargetScan and starBase. According to the prediction, mutant and wild sequences of the binding sites of PDCD4-AS1 and IQGAP2 were designed. The mutant or wild sequence fragments were cloned and conjugated to Promega vectors, designated mut-PDCD4-AS1, wt-PDCD4-AS1, mut-IQGAP2 and wt-IQGAP2, respectively. The vectors were transfected with NC mimic or miR-10b-5p mimic into HEK-293T cells. After 48 h, fluorescence intensity of each group was measured by a dual-luciferase reporter assay system (Promega, WI, USA).