Si circ ccdc66

The cell lines Nthy-ori 3-1, CAL62 and TPC1 were purchased from the American Type Culture Collection (ATCC). All cells were cultured in DMEM (Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Invitrogen; Thermo Fisher Scientific, Inc.) and were places at 37°C in a humidified incubator containing 5% CO₂.
Cell transfection was carried out using Lipofectamine^® 2000 Reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturers' instruction when cells in culture reached 60–80% confluence. Small interfering (si)RNAs targeting circCCDC66 (si-circCCDC66#1, 5′-CAAUUAGAGCAUCAGGAAA-3′; si-circCCDC66#2, 5′-GAGCAUCAGGAAACAGUAC-3′) and negative control (si-NC, 5′-UUCUCCGAACGUGUCACGUTT-3′) were purchased from Shanghai GenePharma Co., Ltd. miR-211-5p mimic and negative control were purchased from Guangzhou RiboBio Co., Ltd (cat. no. miR10000268-1-5). The transfection concentrations of oligonucleotides were as follows: si-NC, 40 nM; si-circCCDC66, 40 nM; miR-211-5p mimic, 50 nM; and miRNA control, 50 nM. Lipofectamine^® 2000 Reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and si-RNAs or miR-mimic were diluted into serum-free DMEM medium, mixed together and incubated for 20 min at room temperature. This solution was subsequently was added to CAL62 and TPC1 cells and transfected for 4–6 h at 37°C in a humidified incubator containing 5% CO₂.