Cell lines and 5-FU
Human GC cell lines BGC-823 and SGC-7901 as well as normal gastric mucosa cell line GES were obtained from the Cell Bank (Shanghai Genechem Co., Ltd., Shanghai, China). All the cell lines have been tested and authenticated by the corporation. 5-FU-resistant cells, BGC823/5FU and SGC7901/5FU, were obtained from Cell Bank of Chinese Academy of Sciences (Shanghai, China). The cells were maintained in RPMI-1640 medium (Gibco-BRL, NY, USA) supplemented with 10% fetal bovine serum (Gibco, NY, USA) at 37 °C and 5% CO2. The anticancer agent 5-FU was purchased from CSNpharm (Chicago, USA).
siRNA synthesis, plasmid construction, and transfection
siRNAs targeting MeCP2 and NADPH oxidase 4 (NOX4) were designed and synthesized by GenePharma Corporation (SGC, Shanghai, China). The negative control was a scrambled sequence siRNA (NC-siRNA). The siRNA sequences are listed in Additional file 1: Table S1. Full-length human MeCP2 complementary DNA was cloned into pCMV2-GV146 vector. pCMV2-GV146-GFP-MeCP2 plasmid (WT), pCMV2-GV146-GFP-Mutation type 1 plasmid (MT1) and pCMV2-GV146-GFP-Mutation type 2 plasmid (MT2) were constructed (Additional file 1: Table S2) . MT1 and MT2 are mutants with defects at two different sites in the MBD domain of MeCP2 so that their proteins cannot bind to the methylated CpG sites of target genes. The reporter plasmid pGL3-NOX4, which contained a 220-bp fragment spanning from 89,323,211 to 89,323,430 located at the promoter region of NOX4, was placed at the downstream of the Firefly Luciferase reporter gene (pGL3-NOX4-luc) (Genechem Co. Ltd., Shanghai, China). BGC-823 and SGC-7901 cells were seeded for 24 h in RPMI-1640 medium without antibiotics, following which siRNAs and/or plasmid vectors targeting specific genes were transiently transfected into cells by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) and cells were cultured for 48 h for subsequent examinations.
RNA extraction and qRT-PCR
Total RNA was extracted from the cell lines and frozen tissues using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. FFPE tissue samples (10 sections) were deparaffinized by incubation for 10 min in xylene and 5 min in 100% ethanol, and were washed with distilled water for 30 s, followed by RNA extraction using the Qiagen FFPE Rneasy Kit (Valencia, CA, USA). The RNA samples were examined spectrophotometrically using Nanodrop (Thermo Fisher Scientific Inc., DE, USA). cDNA was synthesized following the manufacturer's protocol (Takara, Dalian, China). qRT-PCR was conducted using the SYBR Green PCR kit (Takara Biotechnology, Dalian, China). The primers are presented in Additional file 1: Table S3. The qRT-PCR reactions were performed in triplicate for each sample using the IQ5 Multicolor qRT-PCR Detection System (Bio-Rad, USA). β-Actin was used as mRNA control. The 2−ΔΔCt method was adopted for the analysis.
Protein extraction and Western blotting
Total protein was extracted from the tissue samples and GC cells using RIPA buffer (Cell Signaling Technology, Boston, MA) supplemented with protease inhibitors (Roche, Indianapolis, IN, USA). Protein was quantified using the BCA Protein Assay Kit (Pierce Biotechnology, Rockford, USA). Equal amounts of protein lysates were run on 10% SDS-PAGE gels and electro blotted onto polyvinylidene difluoride (PVDF) membranes. The membranes were then blocked with 5% non-fat milk overnight and incubated for 2 h the next day with a 1:1000 dilution of anti-MeCP2 antibody, anti-NOX4 antibody (Santa Cruz Biotechnology), or anti-PKM2 antibody (Abcam). After washing with PBS three times and incubation with appropriate secondary antibody for 1 h, protein visualization was performed by using enhanced chemiluminescence (Pierce Biotechnology, Rockford, USA).
MTT assay for cell viability
Briefly, BGC-823, SGC-7901, BGC-823/5-FU and SGC-7901/5-FU cells were seeded in 96-well plates (5 × 102 cells/well) respectively and cultured at 37 °C for 24 h. The cells were then treated with 5-FU at the concentration of 0, 0.1, 0.5, 1, 5, 10, 20, 40, 80 or 160 μM for 48 h, after which MTT (2.0 mg/ml) was added to each well and the plates were incubated in darkness at 37 °C for 2 h. Subsequently, the medium was removed, formazan crystals were dissolved in DMSO, and optical density (OD) was measured using an ELISA plate reader at 570 nm. The cell viability index was calculated according to the formula: (experimental OD value/control OD value) × 100%.
Colony formation assay
Cells were planted in 6-well plates (1 × 103 cells/well), treated with 10 μM 5-FU for 48 h, transfected with specific si-RNA and/or overexpression plasmid, and incubated in a humidified atmosphere with 5% CO2 at 37 °C for a week. The cell colonies were then fixed with 4% paraformaldehyde for 15 min at room temperature and stained with crystal violet for another 15 min. After washing with PBS, the colonies were recorded by Syngene GBox (Syngene, Cambridge, UK) and only clearly visible colonies with a diameter over 50 μm were counted.
Cell cycle assay
Cells were harvested for analysis by trypsinization 48 h after transfection, washed twice with PBS and fixed with ice-cold ethanol (70%) at 4 °C overnight. After washing twice again, the cells were incubated with Rnase A (0.1 mg/ml) and propidiumiodide (PI, 0.05 mg/ml) for 15 min at room temperature. Then, distribution of cell-cycle stages was detected by flow cytometry (FACS Calibur, BD Biosciences, CA, USA).
Cells were harvested 48 h after transfection, washed twice with PBS and stained using the Annexin V-FITC/PI Apoptosis Detection kit (Invitrogen, Carlsbad, CA, USA). Flow cytometry was performed and the stained cells were counted to quantify cell apoptosis.
Chromatin immunoprecipitation (ChIP)-qRT-PCR
BGC-823 cells were transfected with empty plasmid, WT, MT1 or MT2. ChIP was conducted as previously described . Briefly, BGC-823 cells were cross-linked with 1% formaldehyde and quenching was performed using 125 mM glycine. The chromatin was sonicated into 200-bp (approx.) fragments. Cell lysates were divided into two portions and incubated respectively with 5 μg antibody against IgG or against MeCP2 or GFP (Abcam, Cambridge, MA, USA, Additional file 1: Table S5) overnight at 4 °C. DNA–protein complexes were captured and eluted in TE buffer. After decrosslinking, DNA was extracted using the QIA quick PCR purification kit (QIAGEN, Germany) and analyzed by qRT-PCR using gene-specific primers (Additional file 1: Table S4).
Luciferase reporter assay
BGC-823 cells were seeded into 96-well culture plates (4 wells per group). pGL3-NOX4-luc was amplified in DH5α and was treated with CpG methyltransferase M. Sssl (M0226S, NEB, USA) for 48 h (pGL3-NOX4-luc + Methylation). The cells were transfected with pGL3-luc, pGL3-NOX4-luc, or pGL3-NOX4-luc + Methylation plasmids for 48 h, after which cells (except those transfected with pGL3-luc) were treated with NC-siRNA, MeCP2 siRNA-1/2, null vector, and ov-mecp2 vector, respectively, for 48 h. Then, the luciferase activity per 1000 cells (trypan blue staining) was measured.
Tissue samples of GC and adjacent normal gastric mucosa (5–10 cm away from the primary tumors) of the same patient were collected from 81 patients receiving surgical management at the First Affiliated Hospital of Xi’an Jiaotong University (Xi’an, China) from March, 2017 to January, 2020. The tissues were snapfrozen in liquid nitrogen and stored at − 80 °C. The tissue sections from each patient were reviewed by two experienced pathologists. All patients had not received radiotherapy or chemotherapy before surgery. Follow-up was performed post surgery. The study was conducted following the protocol approved by the ethics committee of the university, and written informed consent was obtained from all patients.
Tumorigenicity assay in nude mice
A total of 12 male, 5-week-old BALB/C nude mice were used to examine tumorigenicity. The animal maintenance and experimental procedures were approved by the Institutional Animal Care and Use Committee of Xi'an Jiaotong University. The mice were divided into three groups (n = 4): sh-Ctrl + PBS, sh-MeCP2 + PBS and sh-MeCP2 + 5-FU. In brief, group one was transplanted with BGC-823/5-FU cells stably expressing sh-Ctrl, while groups two and three were transplanted with cells expressing sh-MeCP2 (1 × 106). Then, 5-Fu at 60 mg/kg of body weight or PBS was injected intraperitoneally one time a week for a total of 5 weeks. At the 35th day, the nude mice were anesthetized through inhalation of 3% isofluorane and given one subcutaneous dose of carprofen (8 mg/kg) for euthanasia. Then xenograft tumors were excised, weighed and pictured. Tumor volume (V) was examined by measuring the tumor length (L) and width (W) and calculated according to the formula: V = (L × W2)/2. The tumor tissues were frozen for qRT-PCR and Western blotting, and embedded in paraffin for immunohistochemistry.
The FFPE tissue samples, including GC patient specimens and xenograft tumor tissues from the nude mice, were sectioned at 4-μm thickness. The sections were deparaffinized with xylene and hydrated with graded alcohol for antibody staining. Then the sections were incubated with primary antibody against MeCP2 (Santa Cruz, CA, USA) at a dilution of 1:200, followed by incubation with secondary antibody. Then, 3,3ʹ-diaminobenzidine (DAB) and hematoxylin were performed. MeCP2 expression was considered high when the proportion of positive cells was over 50% in 5 random fields.
All analyses were performed in the GraphPad Prism program. Data were presented as the mean ± standard error of the mean (SEM) with statistical significance indicated when P < 0.05. Student’s t-test or one-way variance analysis was used for inter-group comparisons.