Cell lines and culture conditions
The normal human gastric epithelial cell line (or called GES-1) was obtained from the Division of Gastroenterology, Department of Medicine, People’s Hospital of Jiangsu Province, China. GES-1 cells were cultured in DMEM (Gibco, America) supplemented with 10% FBS, 2.5 mg/ml amphotericin B, 50 U/ml penicillin, and 50 mg/ml streptomycin. The culture medium was changed every 48–72 h.
Different groups of aspirin induced GES-1 damage
To observe aspirin-induced cell lesion, GES-1 grown to near confluence were cultured in serum-free media containing 0.2% bovine serum albumin (BSA) (Invitrogen, Carlsbad, USA) overnight. Then GES-1 was treated with different concentrations of aspirin (1.25, 2.5, 5 and 10 mmol/L) for 24 h.
GES-1 cells transfection
To investigate the role of MiR-877-5p on GES-1 damage. MiR-877-5p mimic, inhibitor and their oligonucleotides were transfected into GES-1 cells. After cells reached near confluence, then cells were incubated with aspirin at a concentration of 2.30 mmo/L for 24 h. At the end of the experiment, the following assays were performed. All experiments were repeated 3–4 times.
qRT PCR
Total RNA of GES-1 treated with aspirin with and without MiR-877-5p mimic, inhibitor and their oligonucleotides were extracted using Trizol reagent (Invitrogen). The primer sequences used for amplification of genes were as follows: miR-877-5p: 5’-GUAGAGGAGAUGGCGCAGG-3’; U6 snRNA F: 5’-ATTGGAACGATACAGATACAGAGAAGATT-3’, and U6 snRNA R: 5’-GGAACGCTTCAGAATTTG-3’, PDK1 F: 5’-AGGCAAAGGAAGTCCATCT-3’, PDK1 R: 5’-CCCATGCATTGTGCTACC-3’ GAPDH F: 5’-GUAUGACAACAGCCUCAAGTT-3’ GAPDH R: 5’-CUUGAGGCUGUUGUCAUACTT-3’ (Jima, Shanghai, China). qRT-PCR analysis was performed using a standard SYBR-Green PCR kit protocol on a Step One Plus system (Applied Biosystems, Foster City, CA) according to manufacturer's instructions. The relative level of MiR-877-5p and PDK1 transcript was calculated and normalized to GAPDH, with at least 3 repeats per experimental group.
CCK8 Assay
To evaluate aspirin-induced proliferation of GES-1 cells, a CCK8 assay was performed. Cells treated with different concentrations of aspirin (1.25, 2.5, 5 and 10 mmol/L) were seeded in 96well plates at a density of 5 ⋅ 103 cells/well in 200 µl DMEM and cultured at 37˚C for 12 h. Each well was added with 10 ∝l CCK8 reagent (YIFEIXUE Biotechnology, Nanjing, China) and cells were then cultured for another 1–2 h. The cells proliferation was determined by measuring the optical density absorbance at the wavelength of 450 nm.
Flow cytometry analysis
Apoptosis was determined by flow cytometry analysis. A FITC-labeled Annexin V/PI apoptosis assay kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) was employed as previously described[8]. Cells were cultured at room temperature for 5 min using PI (50 µg/ml) solution and 5 µl fluorescein isothiocyanateconjugated AV (17.6 µg/ml), and then 400 µl of binding buffer was added to the cells before detection via flow cytometry assay (FACSCalibur; BD Biosciences, San Jose, CA, USA). Data were analyzed by CellQuest v.5.1 software (BD Biosciences). The experiments were performed in triplicate
GES-1 Transfection
Lipofectamine 2000 mixed with Opti-MEM for 5 min at room temperature, then added respectively pre-mixed miR-877-5p mimic, NC, inhibitor or INC and Opti-MEM for 20 min at room temperature. After GES-1 cells reached 60% confluence, cells were incubated with the mixed medium, medium was changed after 6 h. Total RNA and proteins were harvested at 48 and 72 h after transfection, respectively.
Western Blotting
Total protein was extracted, boiled, and measured with a BCA kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China). After separation with SDS-PAGE gels, protein was transferred onto the nitrocellulose membranes. Then transferred membranes were incubated with primary antibodies (PDK-1 and β-actin at 1:1000 and 1:400 dilutions) at 4 °C overnight, and then secondary antibody labeled HRP was added. Immunoblots were detected by densitometric analysis, and protein strips were analyzed using Image J software (Bethesda, MD, USA).
Prediction and verification of target gene of miR-877-5p
The bioinformatics software was used to predict the target genes of miR-877-5p, and preliminary GO analysis and pathway analysis were performed on these screened target genes.
Statistics
Data are presented as the means ± SE. Statistical significance was determined by unpaired student’s t-test or one-way analysis of variance (ANOVA) followed by the Bonferroni-Dunn post-hoc test. P < 0.05 was considered to be statistically significant. All of the statistical analyses were performed using the Statistical Product and Services Solutions (SPSS) package (Version 20.0, SPSS, Science, Chicago, USA).