Investigating Lung Cancer Cell Lines Using Genetic Manipulations

The human NSCLC lines (NCI-H358, NCI-H1650, NCI-H1568, HCC827, and A549, NCI-H1299) were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). HEK-293 T cell was maintained in DMEM medium (Gibco, USA) and other cells were cultured in RPMI-1640 (Gibco, USA), supplemented with 10% fetal bovine serum (Gibco, USA), and 1% penicillin and streptomycin (Gibco, USA). The cultured cells were maintained at 37°C in a humidified 5% CO₂ incubator. Small interfering RNAs (siRNAs) transfection and plasmid were conducted as described [26 (link)]. When cells reached 40–60% confluence, they were transfected for 48–72 h with siRNAs (circ-in, 5′-GCATCGTGCAGGACTGGAA-3′) targeted to circ_0000677 (constructed by Sangon, Shanghai) at a 50-nM concentration. A scrambled siRNA was utilized as a negative control. Transfection was performed with the lipofectamine 3000 according to the manufacturer’s instructions. To establish stable cell lines overexpressing CCND1, cells were transfected with a pCMV6-CCND1 plasmid DNA (Origene) or the empty plasmid served as the negative control. Human CCND1 cDNA cloned in pCMV6-XL5 was purchased from ORIGENE.