Pmirglo dual luciferase reporter gene vector

The binding sites of miR-124-3p and ANGPTL2 were predicted using the StarBase database (https://starbase.sysu.edu.cn/). Subsequently, the complementary binding sequences and mutated sequences of miR-124-3p and ANGPTL2 were amplified and cloned into the pmiRGLO dual-luciferase reporter gene vector (Promega, Madison, WI, USA) to construct wild-type plasmid pmiRGLO-ANGPTL2-WT and the corresponding mutant plasmid pmiRGLO-ANGPTL2-MUT. In accordance with the instructions of Lipofectamine^TM 2000 (Invitrogen), the plasmid was cotransfected with mimic NC or miR-124-3p mimic (sequence: 5′-UAAGGCACGCGGUGAAUGCCCA-3′, purchased from GenePharma, Shanghai, China) into ovarian granulosa cells KGN (Sunncell, Wuhan, Hubei, China). Afterwards, the luciferase activity was detected 48 h later.

The fragments of AL139294.1 (wild-type and mutant) or 3’-UTR of BRD4 (wild-type and mutant) were amplified and cloned into the pmirGLO dual-luciferase reporter gene vector (Promega, USA). The recombinant vectors were termed lncRNA AL139294.1-WT/MUT and BRD4-WT/MUT, respectively. HEK-293T cells were co-transfected with lncRNA AL139294.1-WT/MUT or BRD4-WT/MUT and miR-204-5p mimics or NC in a 12-well plate. After 48 h of incubation, a dual-luciferase reporter assay system (Promega, USA) was used to measure luciferase activity according to the manufacturer’s instructions. The Renilla luciferase activity was used as a control to normalise the firefly luciferase activity. This experiment was performed in triplicate.