2.2. Cell culture and transfection

The normal thyroid follicular epithelium cell line (Nthy-ori3-1) and PTC cell lines (TPC-1, IHH-4, GLAG-66) were purchased from the American Type Culture Collection (ATCC). The cells were cultured in a medium supplemented with 10% fetal bovine serum (FBS) in RPMI 1640 medium and the conditions of the incubator were 5% CO₂ concentration, 95% humidity, and 37°C. PTC cell was transfection with miR-421 mimic, mimic negative control (NC), miR-421 inhibitor, or inhibitor NC for cell function assay. The transfection reagent was Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA), the liquid was changed 6 hours after transfection, the follow-up experiment was conducted 24 hours later.