Sh circpvt1

Small interfering RNA (siRNA) against circPVT1 and NEK7 (si-circPVT1 and si-NEK7), short hairpin RNA (shRNA) against circPVT1 (sh-circPVT1), miR-181a-5p mimic (miR-181a-5p), miR-181a-5p inhibitor and respective negative controls (si-NC, sh-NC, miR-NC and miR-NC inhibitor) were constructed by GenePharma (Shanghai, China). The sequence of circPVT1 was cloned into the pcDNA basic vector (Invitrogen, Carlsbad, CA, USA) to obtain the overexpression vector pcDNA-circPVT1, and its negative control was marked as pcDNA-NC. These oligonucleotides or vectors were mixed with Lipofectamine 3000 reagent (Invitrogen) and transfected into NSCLC cells with 60% confluence. Harvested cells at different time points were prepared for further experiments.

Small interfering RNA (siRNA) against circPVT1 (si-circPVT1) and its corresponding negative control (si-NC), miR-377 mimic and the control (miRNA NC), miR-377 inhibitor and the control (inhibitor NC), TRIM23 overexpression vector (pcDNA-TRIM23) and the control (pcDNA-Control), and lentivirus-mediated short hairpin RNA against circPVT1 (sh-circPVT1) and its control (sh-NC) were obtained from Genepharma (Shanghai, China). The above plasmids or oligonucleotides were transfected into HCC cells by Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, CA, USA).

TPC-1 cells were purchased from the Chinese Academy of Sciences and cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) in a humidified incubator at 37˚C with 5% CO₂.
miR-384 mimi cs (miR-384; 5'-AUUCCUAGAAAUUGUUCAUA-3'), inhibitors (anti-miR-384; 5'-UAUGAACAAUUUCUAGGAAU-3'), scrambled negative control (NC; 5'-UUCUCCGAACGUGUCACGU-3') oligos miR-NC and anti-miR-NC (5'-CAGUACUUUUGUGUAGUACAA-3'), pGPH1 plasmid containing short hairpin (sh)RNA targeting circPVT1 (sh-circPVT1; 5'-UGGGCUUGAGGCCUGAUCU-3') and pGPH1 plasmid containing scrambled control shRNA (sh-NC; 5'-UUCUCCGAACGUGUCACGUTT-3') were purchased from Shanghai GenePharma Co., Ltd. A total of 5x10⁵ TPC-1 cells was plated in 96-well plates and incubated for 24 h at 37˚C. The plasmids (1 µg/µl), mimics (100 nM) and inhibitors (100 nM) were transfected into TPC-1 cells using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. After 48, the transfection efficiency was assessed.
Stable TPC-1 cells with sh-NC or sh-circPVT1 were selected using 400 ug/ml Geneticin (G418,Thermo Fisher Scientific, Inc.) and 200 µg/ml G418 as the maintenance concentration.