Bioinformatics analysis
Gene expression files of STAD included in TCGA database were accessed and then processed for gene ID transformation using the GTF (GRCh38.p5) files for getting the data of the mRNA expression profile. The profile contains 32 normal samples and 373 tissue samples of gastric cancer. The “edgeR” package in the R language was used for identifying the differentially expressed mRNAs (DE mRNAs) with the critical value set to |logFC|>2 and adj.pvalue < 0.01. Afterwards, the sequences on the upstream 500 bp of the DE mRNAs were applied as putative promoter sequences, which were then used for the extraction of the DE transcription factors (TF) with the JASPAR database (http://jaspar.genereg.net/). The TFs were firstly subjected to the FIMO software (http://meme-suite.org/tools/fimo) for predicting the target mRNAs and then processed for enrichment analysis in DE mRNAs (cor > 0.3, p < 0.05). The TFs with q_value < 0.05 were identified as candidate TFs. Pearson correlation analysis was performed for analyzing the relationship between the target TF and mRNA.
Clinical samples
15 pairs of human gastric cancer tissues and corresponding adjacent normal tissues (margin > 5 cm) from June 2015 to June 2019 were procured from the Zhejiang Provincial People’s Hospital with the approval of all subjects. All cancer samples were pathologically diagnosed and immediately frozen in liquid nitrogen and preserved at -80℃ after being isolated. All subjects had never received any preoperative treatment like chemotherapy or radiotherapy. Our study had been approved by the Ethic Committee of the Zhejiang Provincial People’s Hospital.
Cell culture
Human normal gastric epithelial cell line GES-1 (No: CBP60512) and gastric cancer cell lines AGS (No: CBP60476), SGC-7901 (No: CBP60500), HGC-27 (No: CBP60480) and BGC-823 (No: CBP60477) were all purchased from the Cell Bank of the China Center for Type Culture Collection, Chinese Academy of Sciences (CTCC; Shanghai, China). All cells were cultured in the Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fisher Scientific, Inc., USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and then maintained in a 37 ℃ incubator containing 5% CO2.
Cell transfection
Vectors oe-CTCFL, sh-CTCFL, oe-DPPA2, sh-DPPA2 and their matched negative controls (oe-NC and sh-NC) were synthesized by GenePharma (Shanghai, China). Cells (1 × 105) before transfection were firstly incubated in 12-well plates. LipoFiter assay kit (Hanbio, Shanghai, China) was applied for conducting transfection process per the manufacturer’s protocols. Total RNA and proteins were extracted after 48 h of transfection.
qRT-PCR
Total RNA was isolated from cells using the Trizol (Invitrogen, Carlsbad, USA) and then used for the synthesis of the cDNA with the reverse transcription assay kit (Invitrogen, Carlsbad, USA), following the standard process. qRT-PCR was run on the ABI 7900HT instrument (Applied Biosystems, USA) with the miScript SYBR Green PCR Kit (Qiagen, Germany) under the following thermal cycling conditions: predenaturation at 95℃ for 10 min, 40 cycles of 95℃ for 5 s, 60℃ for 30 s and 72℃ for 2 min. The results were normalized to GAPDH level with the 2−ΔΔCt method. The primers were designed as below:
CTCFL
Forward: 5′-AAAACCTTCCGTACGGTCACTCT-3′;
Reverse: 5′-TGTTGCAGTCGTTACACTTGTAGG-3′;
DPPA2
Forward: 5′-AAGGAGGAGGAGGAGCCAAAC-3′;
Reverse: 5′-TGGTTGGGTGTTTGATTCCAGC-3′;
GAPDH
Forward: 5′- TCCATGACAACTTTGGCATTG-3′;
Reverse: 5′-CAGTCTTCTGGGTGGCAGTGA-3′.
Western blot
RIPA lysate buffer containing 1% protease inhibitor (Beyotime, Shanghai, China) was used for the isolation of total proteins from cells, and the BCA protein assay kit (Beyotime, Shanghai, China) was applied for quantification, according to the manufacturer’s instructions. After being denatured at a high-temperature, the protein samples (30 µg/pore) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred onto the polyvinylidene fluoride (PVDF; Millipore) membranes. 5% skim milk was used to block the membranes for 2 h. Thereafter, the membranes were incubated with primary rabbit polyclonal antibodies overnight at 4℃. The primary antibodies were comprised of CTCFL (ab126766, 1:1000; abcam, China), DPPA2 (ab91318, 1:100; abcam, China) and GAPDH (ab137321, 1:10000; abcam, China). On the following day, the secondary antibody horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG was added onto the membranes for hybridization at room temperature for 120 min. 1 × TBST (Solarbio, Beijing, China) was used to wash the membranes three times. After reaction, the enhanced chemiluminescence (ECL) assay kit (Solarbio, Beijing, China) was employed for the visualization of the protein bands, and then images were captured.
CCK-8
96-well plates were used for cell incubation (200 µl, 1 × 104 cells/ml). At 0, 24, 48 and 72 h, the reagent (20 µg/well) supplied by the cell counting kit-8 (Yeasen) was added into the cells for 4 h of incubation at 37 ℃ in 5% CO2. SpectraMax M5 (Molecular Devices, MD, USA) was used to measure the absorbance values at 450 nm.
Colony formation assay
A measure of 2 ml cell suspension was seeded into 6-well plates at a density of 2 × 102 cells/ml. The mediums were replaced every 4 days. After 3 rounds, the cells were fixed with 4% paraformaldehyde (Invitrogen, China) and then stained in 0.1% crystal violet (Thermo Scientific™ RA Lamb, China). The stained cells were photographed and calculated.
Wound healing assay
Cells (2 ml, 2.5 × 105cells/ml) were inoculated into 6-well plates until the confluence reached 90%. Then the cells were wounded with the tip of a sterile pipette, and sequentially washed with PBS and suspended by the FBS-free mediums at 37℃ in 5% CO2. The wound areas at 0 and 72 h were observed and photographed under an inverted microscope.
Transwell invasion assay
Transwell inserts (sigma, China) that were pre-coated with Matrigel matrix (BD, USA) were put into 24-well plates. 200 µl of cells (1 × 10 5 cells/ml) suspended by FBS-free mediums were planted into the inserts, and 10% FBS-supplemented mediums were added into the plates. After 24 h of incubation at 37℃ in 5% CO2, the cells invaded to the plates were exposed to 4% paraformaldehyde for fixation for 30 min, followed by 0.1% crystal violet for staining for further 30 min. Cells still in the inserts were softly wiped off with a wet cotton swab. Five fields of the view were randomly selected using an inverted microscope, and then photographed for cell count.
Chromatin immunoprecipitation (ChIP)-PCR
The EZ-Magna ChIP assay kit (Millipore) was used for ChIP assay. The specific procedures were as below: 1% formaldehyde solution was used to induce the cross-linking of cells and 140 mM glycine was added for the reaction termination. After the cells were lysed, the nucleoprotein complexes were sheared to the 200–500 bp, and then the obtained DNA fragments were incubated with the antibody for immunoprecipitation overnight at 4℃. Then the samples were washed with 1 × low salt buffer, 1 × high salt buffer, 1 × LiCl buffer and 2 × TE buffer, and sequentially eluted with 200 µl of elution buffer at 37℃ for 15 min. Thereafter, the samples were incubated with 5M NaCl for the reversal of cross-linking overnight at 65℃, and then treated with RNase and protease K. qRT-PCR was performed for identifying the combination of CTCFL and the DPPA2 promoter region.
Statistical analysis
All data were analyzed under the GraphPad Prism 7.0 software (GraphPad Software, Inc., La Jolla, CA). Measurement data were presented as mean ± standard deviation. Comparisons between two groups and among multiple groups were analyzed by Student’s t test and one-way analysis of variance, respectively. Each result was representative of at least three independent experiments. P < 0.05 was set to be a threshold for statistical significance.