Pcdna vector

SNHG16-overexpression vector (SNHG16) and its negative control (Vector), specific small interfering RNA (siRNA) against SNHG16 (si-SNHG16) and siRNA scrambled control (si-NC), miR-424-5p mimic and miR-NC, Anti-miR-424-5p, and Anti-NC were purchased from RiboBio (Guangzhou, China). Construction of SNHG16-overexpression vector (SNHG16) was accomplished by amplifying cDNA of SNHG16 and subcloning into the pcDNA vector (RiboBio). The vectors or oligonucleotides were transfected into T98G or LN229 cells by Lipofectamine 2000 (Thermo Fisher Scientific) in compliance with the manufacturer’s protocol. The primers for SNHG16-overexpression were F 5′-aagcttGCGTTCTTTCGAGG-3′ and R 5′-ggatccTGACGGTAGTTTCCC-3′, including HindIII and BamHI restriction sites. The sequence for si-SNHG16 was 5′-UAAAGACAUGGCACUUUGGGU-3′, and the sequence for si-NC was 5′-UUCUCCGAACGUGUCACGUTT-3′.

A2780 and OVCAR3 cell lines were purchased from Procell (Wuhan, China), and the ISOE80 cell line was obtained from the biotechnology company of Huzheng (Shanghai, China). All cells were cultured in Dulbecco’s modified eagle medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS, Hyclone, South Logan, UT, USA) and 0.1% penicillin/streptomycin (Thermo Fisher Scientific) at 37 ℃ with 5% CO₂.
Overexpression vector of circ-ITCH was acquired by cloning the sequence of circ-ITCH into the pcDNA vector (RiboBio, Guangzhou, China). Small interfering RNAs against circ-ITCH (si-circ-ITCH) and CDH1 (si-CDH1) and their respective controls were synthesized by GenePharma (Shanghai, China). MiR-106a mimics, miR-control, miR-106a inhibitor, and anti-miR-control were synthesized by RiboBio. A2780 and OVCAR3 cells were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).

Chemically synthesized miR-591 mimic, miR-591 inhibitor or miR-negative control (NC), pcDNA-vector and pcDNA-TCF4 were obtained from Guangzhou RiboBio Co., Ltd. (Guangzhou, China). Cells were transfected using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer’s protocol.