2.3. Cell transfection

HTR-8/Svneo cells (5 × 10⁴ cells per well) were cultured in six-well plates overnight and subsequently transfected with control plasmid, control-small interfering (si) RNA (Guangzhou Ribobio Co., Ltd., China), 100 nM inhibitor control (5′-GCCUCCGGCUUCGCACCUCU-3′; Shanghai GenePharma Co., Ltd., China), 100 nM mimic control (5′-UUCUCCGAACGUGUCACGUTT-3′; Shanghai GenePharma Co., Ltd.), WT1-AS plasmid, WT1-AS-siRNA (cat no. siG180524011008-1-5; Guangzhou Ribobio Co., Ltd.; https://www.ribobio.com/product_detail/?sku=siG180524011008-1-5), 100 nM miR-186-5p inhibitor (5′-AGCCCAAAAGGAGAAUUCUUUG-3′; Shanghai GenePharma Co., Ltd.), 100 nM miR-186-5p mimic (5′-CAAAGAAUUCUCCUUUUGGGCU-3′; Shanghai GenePharma Co., Ltd.), or CADM2 plasmid for 48 h, using Lipofectamine^® 2000 reagent (Invitrogen, USA), according to the manufacturer’s protocol. Subsequently, quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to evaluate efficiency of cell transfection.