Cell transfection

A circRap1b overexpression (circRap1b(+)) plasmid and corresponding negative control plasmids were constructed based on the pLO5-ciR-Puro vector (Geneseed Biotech, Guangzhou, China). Hoxa5 and Fam3a overexpression plasmids (Hoxa5(+), concentration: 1150 μg/μL and Fam3a(+), concentration: 1232 μg/μL) were constructed (GenePharma, Shanghai, China). Plasmids encoding siRNAs against Hoxa5, Fam3a, and Kat7 (Hoxa5(–), concentration: 1040 μg/μL; Fam3a(–), concentration: 1182 μg/μL and siKat7, concentration: 1315 μg/μL) and a corresponding negative control (NC) plasmid were constructed by GeneChem (Shanghai, China). HT22-AIS cells were plated in 24-well plates. When the cell confluency reached 70–80%, the cells were transfected with the above plasmids using the transfection reagent Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) and then screened for successful transfection by selection with G428 or puromycin. The transfection efficiency was determined by quantitative real-time polymerase chain reaction and western blot assay.