Human gastric mucosal epithelial cell line GES-1 and gastric adenocarcinoma cell line MKN45 were obtained from the Cell Bank of Type Culture Collection of The Chinese Academy of Sciences. The human gastric epithelial adenocarcinoma cell line AGS and the human kidney 293T cell line were purchased from The American Type Culture Collection. All cell lines were cultured in RPM1640 medium with 10% fetal bovine serum (both Gibco; Thermo Fisher Scientific) and 100 U/ml penicillin and streptomycin (Gibco; Thermo Fisher Scientific) at 37°C, and in a humidified incubator at 10% CO₂.

The Hp GZ7 strain (GenBank accession ID, KR154737.1) was determined to be a typical East Asian strain (CagA⁺) by sequencing in our previous study (16), where it was isolated from clinical gastric cancer tissues with informed consent from the patients and the approval of The Ethics Committee of Guizhou Medical University [Guiyang, China; approval no. 2017(43)]. Hp GZ7 was cultured for 48–72 h at 37°C in a microaerophilic atmosphere on a Columbia agar plate (Thermo Fisher Scientific) containing 10% sheep's blood. Gastric mucosal GES-1, AGS and MKN45 cells were cultured for 24 h in 6-well plates containing antibiotic-free medium and subsequently infected with Hp at a multiplicity of infection (MOI) of 40. The cells were infected for 24 h before they were harvested for western blotting and reverse transcription-quantitative PCR (RT-qPCR). The uninfected cells served as a negative control. All cells were incubated at 37°C and in a humidified incubator at 10% CO₂.

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.

In our previous study (17), Hp NCTC 11637 (ATCC 43504, CagA-positive) was used to intragastrically infect Mongolian gerbils for 24 months to establish Hp-related gastric disease models successfully. In these gerbils, chronic superficial gastritis, erosive gastritis, atrophic gastritis, intestinal metaplasia and well-differentiated gastric cancer were pathologically observed at 3, 6, 12 and 24 months after Hp infection. At different time points, the gerbils (3 gerbils per time point) were sacrificed after anesthesia, and their stomach tissues were removed, fixed and embedded in paraffin (17). In the present study, the paraffin-embedded gerbil stomach tissues of 3 months after Hp infection were cut into 5-µm sections for IHC staining. The animal study was approved by the Animal Care Welfare Committee of Guizhou Medical University (Guiyang, China; approval no. 1900801).

A total of 20 cases of surgically resected gastric cancer and corresponding para-cancerous tissue specimens were collected from patients with gastric adenocarcinoma (13 male and 7 female patients; age, 51.2±9.6 years; range, 40–73 years), who were admitted to the Affiliated Hospital of Guizhou Medical University from March, 2021, to December, 2021. Informed and signed consent was obtained before surgery, and the research protocol was approved by The Ethics Committee of Guizhou Medical University [Guiyang, China; approval no: 2019(19)].

Paraffin-embedded tissue sections were deparaffinized in xylene for three 10 min intervals, incubated in 100% ethanol for three 10 min intervals, and then in a series of graded ethanol solutions (90, 70, 50 and 30% ethanol) for 3 min each. The tissue sections were placed in a boiling pressure cooker for 2.5 min for antigenic repair. After cooling for 10 min in 3% H₂O₂, sections were washed in PBS for 10 min and then incubated with the FTO antibody (1:3,000; cat. no. 27226-1-AP; Proteintech Group, Inc.; Wuhan Sanying) for 2 h at room temperature. Sections were then washed three times with PBS for 10 min intervals and incubated with the goat anti-rabbit IgG antibody labeled with peroxidase HRP (1:10,000; cat. no. SA00001-2; Proteintech Group, Inc.; Wuhan Sanying) at room temperature for 2 h. Sections were then washed three times with PBS for 10 min intervals. A total of 1 ml reagent A and 50 µl reagent B from the DAB Detection Kit (cat. no. ZLI-9018; OriGene Technologies, Inc.) was mixed, two drops were added to the tissue sections for 1–3 min, and then sections were incubated in PBS to stop the reaction. Slides were stained in hematoxylin for 3 min at room temperature, washed in distilled water, stained in ammonia reverse blue solution for 1–2 min, washed in distilled water, dehydrated in a graded ethanol series (50, 70, 90% and absolute ethanol), incubated three times in xylene solution for 10 min intervals, and then mounted. The FTO expression level of each sample was judged according to the number of stained cells and the depth of staining using an optical microscope. Image-Pro Plus 6.0 software (Media Cybernetics, Inc.) was used to calculate the mean integrated optical density of active areas in each group, and the average optical density was taken for analysis.

The total RNA of GES-1 and AGS cells was isolated using TRIzol reagent (Invitrogen; Thermo Fisher Scientific). A Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics, Ltd.) was used for the reverse transcription of RNA according to the manufacturer's instructions. qPCR was next performed using a 2X SYBR Green Master Mix (Roche Diagnostics, Ltd.) in an Illumina Eco™ Real-Time PCR system (Gene Company, Ltd.). The amplification program was as follows: 95°C for 10 min then 38 cycles of 95°C for 12 sec, 60°C for 18 sec and 72°C for 30 sec. GAPDH served as a normalization control, and the 2^−ΔΔCq approach (18) was employed to calculate relative gene expression. Primer 5.0 (PREMIER Biosoft) was used to design primers for qPCR analysis. The FTO primer sequence was 5′-AACACCAGGCTCTTTACGGTC-3′, which was synthesized by Sangon Biotech Co., Ltd.

Total protein of GES-1 and AGS cells was extracted according to the instructions of the high potency RIPA lysate (tissue/cell) research reagent kit (Beijing Solarbio Science & Technology Co., Ltd.). Protein was quantified using a BCA protein quantification kit (Beijing Solarbio Science & Technology Co., Ltd.). For denaturation, 5X loading buffer was added to the protein sample at a 4:1 ratio and boiled for 10 min. A total of 20 µg protein was run on a 10% gel using SDS-PAGE at 80 V for 2 h, proteins were transferred to a polyvinylidene fluoride membrane for another 2 h and the membrane was then blocked with a solution of 20 ml PBS containing 0.05% Tween20 (1X PBST) with 1 g non-fat milk powder at room temperature for 2 h. Incubation with the FTO primary antibody (1:3,000; cat. no. 27226-1-AP; Proteintech Group, Inc.; Wuhan Sanying) was conducted at 4°C overnight. The membrane was washed three times with 1X TBST solution and incubated with the goat anti-rabbit IgG antibody labeled with peroxidase HRP (1:10,000; cat. no. SA00001-2: Proteintech Group, Inc.; Wuhan Sanying) at room temperature for 2 h. Signals were visualized using chemiluminescence ECL (Thermo Fisher Scientific) and imaged using a chemiluminescence imager (Amersham; Cytivia). ImageJ software (version 1.41; National Institutes of Health) was used to quantify the gray value of the image, and the relative protein expression using the ratio of the FTO to HRP-conjugated mouse anti-ACTB monoclonal antibody (1:10,000; cat. no. HRP-60008: Proteintech Group, Inc.; Wuhan Sanying) or HRP-conjugated mouse anti-GAPDH monoclonal antibody (1:10,000; cat. no. HRP-60004: Proteintech Group, Inc.; Wuhan Sanying) was calculated.

The cell culture medium in the confocal dish was discarded and the GES-1 and AGS cells were washed with PBS three times for 5 min intervals. To fix the cells, 1 ml 4% paraformaldehyde was added and incubated for 30 min at room temperature, before washing with PBS three times for 5 min intervals. Cells were permeated with 0.3% Triton X-100 for 50 min at room temperature, before washing with PBS three times for 5 min intervals. The cells were blocked with 5% Bovine Serum Albumin BSA-V (cat. no. A8020; Beijing Solarbio Science & Technology Co., Ltd.) for 1 h at room temperature. The cells were incubated with FTO primary antibody (1:50; cat. no. 27226-1-AP; Proteintech Group, Inc.; Wuhan Sanying) overnight at 4°C and then washed with PBS three times for 5 min intervals. CoraLite594-conjugated goat anti-rabbit IgG(H+L) (1:200; cat. no. SA00013-4; Proteintech Group, Inc.; Wuhan Sanying) was added to the cells and incubated for 1 h at room temperature. Then, 100 µl of Antifade Mounting Medium with DAPI (cat. no. P0131-25 ml; Beyotime Institute of Biotechnology) was added to each well in turn. The cells were carefully covered with a slide and imaged by a laser confocal microscope. Image fluorescence intensity was quantified using ImageJ software (version 1.41; National Institutes of Health).

FTO RNA-seq V2 mRNA expression data from specimens in the gastric cancer dataset (including 408 gastric cancer specimens and adjacent tissues) were downloaded from the TCGA database (https://tcga-data.nci.nih.gov/tcga/) and preprocessed. From these, 211 specimens were screened for sex, age, tumor size (T1, T2, T3 and T4), lymph node metastasis (N0, N1 and Nx), distant metastasis (M0, M1 and Mx) and American Joint Committee on Cancer (AJCC) pathological feature stage (AJCC stage I + II, III + IV) (19). A total of 378 cases from TCGA database were selected as the gastric cancer group. In addition, according to the level of FTO mRNA expression, cases in the gastric cancer group were divided into the high expression group and the low expression group according to the median value, with 180 cases in each group.

To analyze the genes that were positively and negatively correlated with FTO expression the LinkedOmics database (http://linkedomics.org/login.php) was used. The CANCER COHORT was selected as ‘TCGA_STAD’, the SEARCH DATASET as ‘HiSeq RNA’, the SEARCH DATASET ATTRIBUTE as ‘FTO’, the TARGET DATASET as ‘HiSeq RNA’ and the STATISTICAL METHOD as ‘Pearson Correlation test’. Gene function enrichment analysis was performed using the STRING website (https://cn.string-db.org/). The website operation steps were followed to carry out Gene Ontology (GO) Enrichment analysis for ‘Molecular Function’, ‘Biological Process’ and ‘Cell Component’.

A total of 1×10⁶ AGS and MKN45 cells were cultured in a 6-well plate overnight. When the degree of confluence reached ~90%, a horizontal cell scraping strip of equal width (~500 µm) was drawn through the center of the well, and the wounded cells were washed with PBS and incubated for a further 48 h in the medium with 4% FBS. Imaging was conducted using an optical microscope at 0 and 48 h to measure the cell damage. The migration ability of cells was evaluated by comparing the relative wound width at 48 h with the wound width at 0 h. The relative scratch healing rate was calculated as follows: Percentage relative scratch healing rate=[gap distance (T0-T48)/gap distance at T0] ×100%, with T0 and T48 corresponding to the width of the wound at 0 and 48 h, respectively.

Invasion assays were performed in Transwell plates (Costar; Corning, Inc.) precoated with Matrigel (BD Bio sciences). The chamber coating was performed at 37°C for 1 h. A total of 2×10⁴ of each cell line (AGS and MKN45) diluted in serum-free DMEM medium was added to the upper chamber and 500 µl DMEM containing 10% FBS was added to the lower chamber as a nutritional attractant. After incubating for 48 h at 37°C, the cells inside the upper chamber were removed and the cells invading through the membrane were fixed with 4% paraformaldehyde for 30 min at room temperature. The fixed cells were then stained with 0.1% crystal violet for 30 min at room temperature and counted under an optical microscope.

The SPSS 18.0 software (SPSS, Inc.) was used for data analysis. Unpaired t-test was used to compare the two independent Hp infected and uninfected groups. To analyze the differences in FTO expression between gastric cancer tissues and paired adjacent tissue, paired t-test was used. One-way ANOVA followed by Dunnett's post hoc tests was used when multiple groups were compared respectively with control groups. One-way ANOVA followed by Tukey's post hoc test was used to compare all pairs of columns containing multiple groups. The following settings for GEPIA were used as: ‘Expression on Box Plots’, ‘Gene as FTO’, ‘|log2FC| Cutoff as 1’, ‘P-value Cutoff as 0.01’, ‘Datasets as STAD’, ‘Log Scale as log2(TPM + 1)’, ‘Jitter Size as 0.4’ and ‘Matched normal date’ as ‘TCGA normal and GTEx data’. Survival curve analysis was performed using the Kaplan-Meier method and the log-rank test using GEPIA data. To analyze the relationship between FTO expression and clinicopathological parameters, χ² test was used. Spearman's correlation test was used for analysis of FTO-related gene screening and Fisher's exact test was used for gene function enrichment analysis. P<0.05 was considered to indicate a statistically significant difference.

Of the cell lines used, the cell lines with lowest FTO expression were the AGS and GES-1 cell lines. Therefore, these two cell lines were selected for follow-up experiments (Fig. 1A). Following GES-1 and AGS infection with the Hp GZ7 strain at MOI 40:1 for 24 h, FTO mRNA and protein levels were detected by RT-qPCR and western blotting, respectively, and both the mRNA and protein levels in infected cells were significantly higher than those in uninfected cells (Fig. 1B and C). Immunofluorescence demonstrated that FTO was mainly expressed in the cytoplasm of gastric epithelial cells, and the total fluorescence intensity of FTO in Hp infected cells increased (Fig. 1D). The expression level of FTO protein in the gastric tissues of Mongolian gerbils infected with Hp was also significantly higher than that of the control group (Fig. 1E).

A total of 20 gastric cancer specimens and 20 corresponding adjacent tissue sections were examined by immunohistochemistry to determine the differences in FTO expression levels. Positive staining of FTO protein was visualized as brown-yellow granules, mostly located in the cytoplasm and nucleus (Fig. 2A). The protein level of FTO in gastric cancer tissues was also significantly higher than that in the adjacent tissues (Fig. 2A). FTO mRNA expression data based on a total of 408 gastric cancer cases and 211 normal tissues were available from the TCGA database. Bioinformatics analysis indicated that the mRNA expression of FTO in gastric cancer was significantly higher than that in normal tissues (Fig. 2B). Kaplan-Meier survival curves were used to explore the prognostic value of FTO expression on the overall survival rate of patients with gastric cancer. The overall survival rate of patients with high FTO expression was significantly lower than that of patients with low FTO expression (Fig. 2C).

The gastric cancer group included 242 males and 136 females; 118 cases aged ≤60 years and 260 aged >60 years; 102 cases of T1+T2 stage and 276 of T3+T4 stage; 121 cases of N0 stage, 99 of N1 stage, 81 of N2 stage, 72 of N3 stage and 5 of Nx stage; 334 cases in M0 stage, 26 in M1 stage and 18 in Mx stage; 182 cases in Stage I+II and 196 in Stage III+IV. The χ² test was used to examine the relationship between FTO mRNA expression and the clinicopathological characteristics of patients with gastric cancer in the TCGA database. The results demonstrated a positive association between the level of FTO mRNA expression and clinicopathological parameters in patients with gastric cancer, such as T stage, M stage and AJCC stage, and a high expression level of FTO mRNA was associated with higher T stage, M stage and AJCC stage. There was no association between FTO mRNA expression level and sex, age or N stage of patients (Table I).

The LinkedOmics database was used to analyze genes related to FTO in patients with gastric cancer, based on Spearman's correlation test. The FTO gene in gastric cancer was positively correlated with 16,601 genes, including ZFP90, chromodomain helicase DNA binding protein 9 (CHD9) and protocadherin γ subfamily B7 (PCDHGB7) (Fig. 3A), and was negatively correlated with 3,623 genes, including halo acid dehalogenase like hydrolase domain containing 3 (HDHD3) and estrogen related receptor α (ESRRA) (Fig. 3B). GO enrichment demonstrated that RNA stability and RNA metabolism were the biological processes in which FTO related genes were most significantly enriched during gene expression regulation (Fig. 3D). The molecular functions of FTO-related genes that were most significantly enriched were oxidative RNA demethylase activity and mRNA binding. The cellular components most significantly enriched were the RNA m6a methyltransferase complex and nuclear speckles. The enrichment results of FTO-related genes suggest a possible function for FTO in cell proliferation and metabolism and a role in the tumorigenesis and progression of gastric cancer.

Lentiviral expression vectors were used to establish stable AGS and MKN45 cell lines containing a knocked down FTO gene. Western blot analysis demonstrated that FTO was successfully knocked down in AGS and MKN45 cells, which was rescued by Hp infection (Fig. 4A). Hp infection of cell lines with or without a knocked down FTO gene were used to investigate the role of FTO on Hp-induced cell migration and invasion abilities by wound healing and Transwell assays. The results demonstrated that Hp enhanced cell migration and invasion abilities in AGS and MKN45 cells (Fig. 4B and C). However, the migration and invasion abilities of cells were reduced in cell lines where the FTO gene had been knocked down. Altogether, these findings suggested that knocking down the FTO gene may inhibit the promoting effect of Hp on the migration and invasion ability of gastric cancer cells.