Lentivirus

The CBP/PAG gene sequence was designed by Dr Wang in our laboratory. Lentiviruses containing CBP and enhanced green fluorescent protein (EGFP) genes, and lentivirus containing EGFP alone genes were constructed by Shanghai GeneChem Co., Ltd. (Shanghai, China). Briefly, Jurkat cells were plated at 5,000 cells/well in 96-well plates 24 h prior to transfection. The Lentiviruses [10⁸ TU/ml; multiplicity of infection (MOI) is 50] and polybrene (Shanghai GeneChem Co., Ltd., Shanghai, China; 0.5 mg/ml; 10 µl/well) were added to the cell suspension, which was then cultured in at 37°C in a 5% CO₂ incubator. At 8 h post-transfection, target cells were centrifuged at 200 × g for 10 min at 37°C. Subsequently, cells were cultured in RPMI-1640 medium containing 10% FBS and 1% penicillin and streptomycin at 37°C with 5% CO₂. At 48 h post-infection, cells were visualized using a fluorescent confocal microscope (FV-1000; Olympus Corporation, Tokyo, Japan) and single-cell sorted using a Guava EasyCyte mini flow cytometer (EMD Millipore). EGFP fluorescence (excitation at 488 nm) was detected using a 530/30-nm bandpass filter. Single cell sorting allowed the visualization of green fluorescence in microcultures.

For the construction of stable overexpressing and stable knockdown of GC cell lines, we purchased lentivirus from Genechem (Shanghai, China) and infected MGC803 and AGS with lentivirus. Subsequently, 10% FBS containing 2 μg/mL puromycin (Beyotime, Shanghai, China) was used to screen stably overexpressing and stably knockdown GC cell lines.
circRAB31 siRNA, all miRNA mimic, inhibitor, and their corresponding normal control were designed and synthesized by RiboBio (RiboBio, Guangzhou, China). The detailed nucleotide sequences are listed in Table S1. WT and MUT dual-luciferase reporter plasmid of circRAB31 and 3′UTR of PTEN were bought from Gene Create (Gene Create, Wuhan, China). For transfection, Plasmid and oligonucleotide were co-transfected into the GC cell lines with Lipofectamine 2000 (Invitrogen, Carlsbad, CA).

In this experiment, miR-142a-3p overexpression, knockdown and control groups were constructed using the plasmid hU6-MCS-Ubiquitin-EGFP-IRES-puromycin (Genechem, Shanghai, China). Lentivirus (Genechem) was used to package the plasmid, and finally used to transfect cells. The method is as follows: the vector and the target fragment were each digested with the same restriction enzymes (Beyotime, Shanghai, China) and, after agarose electrophoresis, the gel was cut to recover the product. The recovered vector and the target fragment were ligated overnight at 16°C; the ligation product was used to transform Escherichia coli. The Plasmid Midi Preparation Kit (Beyotime) was used to extract the plasmids.
The cells were planted in a 24-well plate at a density of 30–50%, and GM, lentiviral infection enhancing reagent (Genechem) and 1.5 × 10⁷ TU/mL Lentivirus were added sequentially. The total volume was 500 μL. After 72 hours of culture, the culture medium was replaced with GM and the transfection efficiency was observed under a fluorescence microscope (MZ75, Leica, Bensheim, Germany). Cells expressing green fluorescent protein were deemed to be successfully transfected. The cell transfection rate was calculated as the number of green fluorescent cells/total cells.