The human LSCC cell lines TU177, TU686, TU212, LSC-1 and Hep-2 were obtained from Shanghai Institute of Biological Science Cell Center. Hep-2 cells were cultured in F12K medium (Invitrogen), and the remaining cells were cultured in RPMI 1640 medium (Invitrogen).
Cell transfection was carried out when cells in culture reached 60–80% confluence using Lipofectamine® 2000. Small interfering RNAs (siRNAs) targeting circZNF609#1 (si-circZNF609#1, 5ʹ-GTCAAGTCTGAAAAGCAATGA-3ʹ), circZNF609#2 (5ʹ-TGCCCTAGTACTACCCTGCAT-3ʹ) and circZNF609#3 (5’-TTGACTGCATCGTAGCCAAAC-3’) and negative control (si-NC, 5’-UUCUCCGAACGUGUCACGUTT-3’) were purchased from Shanghai GenePharma Co., Ltd. The miR-134-5p mimetic, agomir and controls were purchased from Guangzhou RiboBio Co., Ltd. The transfection concentrations of oligonucleotides were as follows: si-NC, 40 nM; si- circZNF609, 40 nM; si-NC, 40 nM; si-EGFR, 40 nM; miR-134-5p mimetic, 50 nM; and miRNA control, agomir and miRNA control, 50 nM. Lipofectamine® 2000 Reagent and si-RNAs or miR-mimic were diluted with serum-free DMEM medium, mixed together and incubated at room temperature for 20 min. This solution was subsequently added to LSC-1 and Hep-2 cells for transfection at 37°C for 4–6 h in a humidified incubator containing 5% CO2.
Cell transfection was carried out when cells in culture reached 60–80% confluence using Lipofectamine® 2000. Small interfering RNAs (siRNAs) targeting circZNF609#1 (si-circZNF609#1, 5ʹ-GTCAAGTCTGAAAAGCAATGA-3ʹ), circZNF609#2 (5ʹ-TGCCCTAGTACTACCCTGCAT-3ʹ) and circZNF609#3 (5’-TTGACTGCATCGTAGCCAAAC-3’) and negative control (si-NC, 5’-UUCUCCGAACGUGUCACGUTT-3’) were purchased from Shanghai GenePharma Co., Ltd. The miR-134-5p mimetic, agomir and controls were purchased from Guangzhou RiboBio Co., Ltd. The transfection concentrations of oligonucleotides were as follows: si-NC, 40 nM; si- circZNF609, 40 nM; si-NC, 40 nM; si-EGFR, 40 nM; miR-134-5p mimetic, 50 nM; and miRNA control, agomir and miRNA control, 50 nM. Lipofectamine® 2000 Reagent and si-RNAs or miR-mimic were diluted with serum-free DMEM medium, mixed together and incubated at room temperature for 20 min. This solution was subsequently added to LSC-1 and Hep-2 cells for transfection at 37°C for 4–6 h in a humidified incubator containing 5% CO2.