Si egfr

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.

The miR-335 inhibitor, miR-335 mimic, si-EGFR and corresponding controls were synthesized from RiboBio (Guangzhou, China). The pcDNA-EGFR and pcDNA were provided by GenePharma (Shanghai, China).
Before transfection, HUVECs were inoculated in 100 mm diameter dishes for 24 h. Then, according to the manufacturer's standard procedure, the miR-335 inhibitor, miR-335 mimic, si-EGFR, pcDNA-EGFR and their corresponding controls were transfected into HUVECs using Lipofectamine™2000 (Invitrogen). Each assay was repeated three times independently.