Lentivirus-Mediated Overexpression

The pCMV-MCS-PGK-Puro vector (PHY-008, HanYin Biotech, Shanghai, China) was used for the stable overexpression of LINC02487. This vector contains a puromycin resistance gene and is introduced into cells using lentiviral transfection. Full-length LINC02487 was synthesized, amplified, and cloned into the pCMV-puro lentiviral vector (HanYin Biotech). pCMV-puro empty-vector transduction particles were used as controls. Cells were seeded in a 6-well plate at a density of 6×10⁵ cells/well 24 h before infection. The medium was removed, and 10–20 multiplicity of infection (MOI) of MISSION pCMV-LINC02487-puro lentiviral particles and 2 mL of Opti-MEM medium (Gibco) supplemented with polybrene (8 μg/mL, Sigma, St. Louis, MO, USA) was added to the cells. Lentiviral infection was performed at 37°C for 6 h unless intensive cell toxicity was observed. The medium was then replaced with 2 mL of fresh medium. Infected cells were allowed to grow for 48–72 h and then selected with puromycin-containing medium (Sigma). The expression of LINC02487 was confirmed by RT-qPCR.