The BA-induced IM cell model was described previously . GES-1 and AGS (purchased from ATCC) were cultured in PRIM-1640 medium (Gibco, USA) with 10% fetal bovine serum (Gibco, USA), 100 mg/ml streptomycin and 100 U/ml penicillin. The primary cells were incubated in ICell Primary Epithelial Cell Culture System (icellbioscience, China) with 2% fetal bovine serum (Biocytosci, USA). Deoxycholic acid (DCA) was chosen for the following experiments as it is a major hydrophobic bile acid with potent cytotoxicity (BiocytoSci, USA).
Tissue microarray and human IM samples
A normal tissue microarray containing 24 cases (BN01011b) and a gastric IM tissue microarray (ST8017a) containing 80 cases of gastric IM cases were purchased from Alenabio Biotech (China). Ten paired human gastric IM specimens were obtained from patients who underwent gastroscopy. The pathological data of these specimens was collected from the Department of Pathology. Our study was approved by the ethics committee of Xijing Hospital. All patients signed informed consent before the specimens were obtained. To exclude the effect of Hp infection, all of the patients we selected were Hp negative.
Immunohistochemistry (IHC) was performed using anti-FXR (1:50, abcam, #187735), anti-SNAI2 (1:50, Invitrogen, #PA5-73015) antibodies following the manufacturer’s instructions. The IHC results were independently evaluated by two professional pathologists independently. The scoring was the product of positive rate and intensity of staining. Staining intensity: negative (0), weak (1), moderate (2), and strong (3). The positive rate of staining: <10% (0), 10%-25% (1), 26%-50% (2), 51%-75(3), and >75% (4).
Cells were plated in 4-well chamber slides (Millipore, USA). Then, the cells were washed by twice with cold PBS, fixed with 500μl of 4% paraformaldehyde for 35 minutes, permeabilized with 200μl 1% Triton X-100, for 10 minutes and blocked with 500μl of goat serum, for 30 minutes. Then the cells were incubated with primary antibodies at 4°C overnight. Then the cells were incubated with the FITC secondary antibody at room temperature for 2 hours. Finally, the nuclei were stained with DAPI (Solarbio) for 10 minutes. IF staining for FXR was performed in GES-1 cells. The primary antibodies were a rabbit anti-human FXR antibody (1:100, abcam, #187735).
RNA extraction and real-time PCR
TRIzol® reagent (Invitrogen, USA) was used to extract the total RNA from cell lines and human tissue samples according to a standard protocol. Then RNA was reverse transcribed into cDNA using the PrimeScript® RT Reagent kit (Takara Biotechnology, Japan) at 37˚C for 15 minutes and 85˚C for 5 seconds, followed by temperature maintenance at 4˚C. And qPCR was conducted using SYBR Premix Ex Taq II (Takara Biotechnology, Japan) on a CFX96™ Real-Time PCR Detection system (Bio-Rad Laboratories, USA). β-actin and U6 were used as the internal control and the 2∆∆Cq method was utilized to quantify the relative mRNA expression of each gene. Sequences of gene-specific primers are shown in Table 3.
Protein extraction and western blot analysis
Total protein was extracted by mixing cells with radioimmunoprecipitation assay (RIPA) buffer (Beyotime, China), which was supplemented with protease and phosphatase inhibitors. Western blot analysis was carried out following standard procedures. The following antibodies were used for this experiment: anti-FXR (1:200, abcam, #187735), anti-SNAI2 (1:1000, abcam, #106077), anti-HDAC6 (1:1000, Cell Signaling Technology, #7558), anti-HNF4α (1:2000, Abcam, #92378), anti-CDX2 (1:1000, Cell Signaling Technology, #12306), and anti-β-actin (1:5000, Bioworld, #AP0060). The primary antibody dilution buffer, PREstain Protein Ladder and Western SuperSensitive Substrate were purchased from BioCytoSci (USA).
Synthetic miR-1 agomir, the corresponding negative control oligonucleotides and plasmid small interfering RNA (siRNA) were purchased from Genepharma (China), and the sequence of them was shown in Table 4. Attractene transfection reagent was added to Opti-MEM to generate the transfection medium. Cells were cultured in the transfection medium and it was replaced with fresh medium after 6 hours. Then the cells were grown in 5% CO2 at 37°C for an additional 48 hours. The transfection regent was purchased from Thermo Fisher Scientific (USA) and was used following the manufacturer’s protocol. SNAI2 and FXR overexpression lentiviral vector were designed and provided by Genechem Co. Ltd. (China). GES-1 cells were infected with the lentivirus for 48 hours and then cultured with 2 μg/ml puromycin for 7 days.
Dual-luciferase report assay
We amplified a specific sequence upstream of the transcription start sites of the SNAI2 promoter by PCR, and then cloned it into a pGL3-basic luciferase vector (Invitrogen, USA) to generate SNAI2-p1 (-2000bp~+500bp), SNAI2-p2 (-543bp~+500bp), SNAI2-p3 (+398bp~+500bp) and SNAI2-p4 (+415bp~+500bp). We also amplified the 2000 bp sequence upstream of the transcription start sites of the miR-1 promoter, and then cloned it into the pGL3-basic luciferase vector.
GES-1 cells were transiently transfected with a miR-1 promoter vector. Then, ChIP analysis was performed according to the standard method of the Magna ChIP G Assay kit (Millipore, USA). Chromatin was immunoprecipitated with anti-FXR, anti-SNAI2 or the negative control IgG. Finally, immunoprecipitated DNA-protein complexes were isolated and a real-time PCR assay was carried out to examine the quantity of specific proteins. The primers for the SNAI2 and miR-1 promoters are listed in Table 3.
The SPSS software (V.19.0, SPSS, USA) statistical package was used to conduct all statistical analyses. All continuous data are expressed as the mean±SD. The χ2 test was used to compare the frequencies of categorical variables. Mutual associations among clinical results were assessed by using Spearman’s rank correlation. Statistical comparisons between two groups were analyzed with unpaired Student’s t test. P values less than 0.05 were considered statistically significant.