The potential target sites for miR-449a on the mouse SCN2B mRNA 3′-UTR seed regions, including both wild-type (wt) and mutant (mut) sequences, were cloned. The artificially cloned sequences were inserted downstream of the luciferase reporter gene, pGL3 (Guangzhou RiboBio Co., Ltd.), to generate the SCN2B 3′-UTR-wt and SCN2B 3′-UTR-mut vectors, as described previously (40 (link),41 (link)). Briefly, 293T cells (purchased from Institute of Biochemistry and Cell Biology, Shanghai, China) were seeded in 96-well plates and co-transfected with 100 ng/ml of each pGL3-SCN2B 3′-UTR-wt or -mut vector and 35 nM miR-449a mimics or NC (Guangzhou RiboBio Co., Ltd.). Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) was used as the transfection reagent. The effects of miR-449a treatment on luciferase activity were measured at 48 h post-transfection. The firefly and Renilla luciferase activities were separately measured using a Dual Luciferase Reporter Assay System kit (Promega Corporation) and a Tecan M200 luminescence reader (Tecan Group, Ltd.) according to the manufacturer's instructions. The relative transcriptional activity was normal-ized to Renilla luciferase activity.