The sequence of short hairpin FTO (shFTO) was 5′-CACCAAGGAGACTGCTATTTA-3′ and that of nonsense shRNA (sh-NC) was 5′-GCUUCGCGCCGUAGUCUUA-3′. In the design of shFTO and sh-NC, restriction sites (BamHI and EcoRI) and a loop at both the 5′ and 3′ ends and in the middle of the sequence were added. For plasmid construction, the shRNA was added to a 0.2 ml centrifuge tube, placed in a PCR machine at 95°C for 5 min, and then allowed to stand at room temperature for 20 min to form a double-stranded oligo fragment. The psi-LVRU6GP vector (Guangzhou Fulengen Co., Ltd.) was digested with BamHI and EcoRI at 37°C for 1 h, fragments were separated by 0.8% agarose gel electrophoresis, and were then purified using a gel recovery kit (Tiangen Biotech Co.). The double-stranded oligo fragment was mixed with the digested vector, and the ligase reaction was carried out at 16°C overnight with T4 DNA ligase. The ligated product was transformed into competent Escherichia coli DH5α cells and positive clones were selected for using ampicillin. Plasmid DNA was extracted by DNA plasmid extraction kit (Tiangen Biotech Co.) for sequencing and identification. 293T cells were cultured until 80% confluency and then transfected with 2 µg shFTO plasmid or empty vector plasmid and lentivirus package plasmids psPASX2 (1.5 µg) and PMD2G (0.5 µg) at the ratio of 4:3:1 in the presence of 5 µl H4000 (Engreen Biosystem Ltd.). After 48 h of transfection, the virus supernatant was extracted and used to infect AGS and MKN45 cells at an MOI of 40. When the AGS and MKN45 cells reached 90% confluency, puromycin (final concentration, 1 µg/ml) was added and stable cell lines with knocked down FTO expression were isolated after 1 week.