Transfection and Selection of Stable Cell Lines

HGC-27 cells seeded in 6-well plates were transfected with 2 μg shRNAs against lncHEIH or scrambled shRNAs using Lipofectamine 3000 (Invitrogen) in Opti-MEM serum-free medium according to the manufacturer’s protocol, and stably transfected cells were selected with puromycin (1.5 μg/ml) for 2 weeks. For siRNAs transfection, the cells were plated in a 12-well plate at a density of 1 × 10⁵ cells/well and transfected 50 nmol/L siRNA into cells using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. Experiments were routinely performed in triplicate wells and repeated at least three times. The siRNAs were obtained from Sangon Biotech, Co., Ltd. (Shanghai, China). The sequences of siRNAs and shRNAs were as follows: sense of si-lncHEIH1 5′-GCGCCUUCCCUCUAACCUUAA-3′; sense of si-lncHEIH2 5′-GGCAAGAUGAACGUCUGAAAU-3′; sense of si-EZH2-1 5′-GAUAGAGAAUGUGGGUUUA-3′; sense of si-EZH2-2 5′-GGAUGGUACUUUCAUUGAA-3′; shRNA-HEIH-1: 5′-TGCGCCTTCCCTCTAACCTTAATT CAAGAGATTAAGGTTAGAGGGAAGGCGCTTTTTTC-3′; and shRNA-HEIH-2: 5′-TGGCAAGATGAACGTCTGAAA TTTCAAGAGAATTTCAGACGTTCATCTTGCCTTTTTTC-3′.