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.