MG-63 cell culture and transfection

Human OS MG-63 cells, purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China), were cultured in minimum essential medium/Earle's balanced salt solution supplemented with 10% fetal bovine serum, 50 U/ml penicillin and 50 mg/ml streptomycin (all Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) at 37°C in a humidified 5% CO₂ atmosphere. Cells were plated onto 6-well plates (5×10⁵ cells/well) and the medium was replaced every 3 days. Once the cells reached 70% confluence, 100 nM miR-499a-5p mimic (forward 5′-GACACGGCTCCGTCAC-3′; reverse 5′-ATCCCTGATCAACTGTCCGCC-3′) or its negative control (miR-499a-5p mimic NC) (forward 5′-AACCAGGCCTCCACCG-3′; reverse 5′-GCCACGCATGTCTTATACTGC-3′) (Sangon Biotech Co., Ltd., Shanghai, China) was transfected into the MG-63 cells using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. After 4–6 h co-transfection, the transfection reagent was exchanged with fresh normal medium, and 24 h later, total RNA or total protein was extracted.