Cell culture, transfection, and lentiviral infection
Two human OS cell lines (MG63 and U2OS), a normal human fetal osteoblastic cell line (hFOB1.19) and a human embryonic kidney cell line (HEK293T) used in this study were purchased from Chinese National Collection of Authenticated Cell Cultures. MG63 cells were cultured in DMEM medium (Hyclone, USA) supplemented with 10% fetal bovine serum (HyClone, USA) and 1% penicillin/streptomycin (Invitrogen, USA) at 37 °C under 5% CO2 and saturated moisture. U2OS cells were cultured in McCoy’s 5a medium (Hyclone, USA) supplemented with 10% fetal bovine serum (HyClone, USA) and 1% penicillin/streptomycin (Invitrogen, USA) at 37 °C under 5% CO2 and saturated moisture. hFOB1.19 cells were cultured in DMEM/F12 (1:1) medium (Hyclone, USA) supplemented with 10% fetal bovine serum (HyClone, USA) and 1% penicillin/streptomycin (Invitrogen, USA) at 37 °C under 5% CO2 and saturated moisture. HEK293T cells were cultured in DMEM/high glucose medium (Hyclone, USA) supplemented with 10% fetal bovine serum (HyClone, USA) and 1% penicillin/streptomycin (Invitrogen, USA) at 37 °C under 5% CO2 and saturated moisture. The authenticity of cell lines was verified by DNA fingerprinting before use. MiR-204-5p and its negative control (miR-NC), RUNX2 eukaryotic expression recombinant pIRESpuro2-RUNX2 were transfected transiently into OS cells using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s instructions. To prepare sh-circ_0001722 lentiviral particles, the lentiviral vector harboring sh-circ_0001722 full hairpin sequence and packaging vectors were transfected into HEK293T cells using iMFectin Poly DNA Transfection Reagent (GenDEPOT, USA) following the manufacturer’s suggested protocols. The transfection medium was changed at 8 h after transfection and then cells were cultured for 36 h. The lentiviral particles were harvested by filtration using a 0.45 µm sodium acetate syringe filter and then combined with 8 μg/ml of polybrane (Millipore, USA) and infected overnight into 60% confluent OS cells. The cell culture medium was replaced with fresh complete growth medium and after 24 h, cells were selected with 2 μg/ml of puromycine for an additional 24 h. The selected cells were used for experiments. MiR-204-5p mimic and its negative control (miR-NC), pIRESpuro2-RUNX2 vectors, lentiviral vectors harboring sh-circ_0001722 full hairpin sequence and mock sequence, were purchased from Shanghai GenePharma Co., Ltd.