Patients and tissue specimens
Gastric tissues were collected from patients from the First Affiliated Hospital of Anhui Medical University from July 2017 to June 2018. A total of 150 subjects were enrolled in this study, including 30 healthy subjects who underwent routine endoscopic screening for gastric cancer, 90 patients with atrophic gastritis associated with various degrees of dysplasia (mild, moderate and severe, n = 30 in each group) and 30 patients with early gastric cancer. All specimens were histopathologically confirmed. This study was approved by the Human Research Ethics Committee of Anhui Medical University. Written informed consents were obtained from all participants.
Immunohistochemistry
Paraffin sections were baked overnight and routinely processed. Endogenous peroxidase activity was quenched by 3% hydrogen peroxide for 10 minutes. Then, the antigen were then blocked for 20 minutes with Bovine serum albumin (BSA, 5%) and the slides were incubated with antibody at 4°C overnight. Finally, the tissues were incubated with SABC working solution and visualized with 3,3-diaminobenzidine (DAB). The images were acquired under the microscope (Nikon, Japan) at 200× magnification.
Cell culture and transfection
The human gastric cancer cell line SGC–7901 was obtained from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI–1640 Medium (Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Life Technologies) at 37°C in a humidified atmosphere with 5% CO2. pcDNA3.1/PDCD4 plasmid was kindly provided by Dr. Qian-ben Wang (University of Duke, USA). Lipid-mediated transfection was used to obtain PDCD4 high-expressed SGC–7901 cell line according to the manufacturer’s protocol.
Western blot
After the protein was processed, the protein liquid was separated by SDS–PAGE and transferred into nitrocellulose membranes (Millipore, Bedford, MA). The blot was blocked in blocking buffer (5% not-fat dry milk and 1% Tween–20 in PBS) for 2 hours at room temperature, and then incubated with appropriate primary antibodies in blocking buffer overnight at 4oC. The membranes were washed and incubated with secondary antibodies and were analyzed with an enhanced chemiluminescent reagent. The bands from western blotting were quantified by Quantity One analysis software (Bio-Rad).
Immunofluorescence
The cells were fixed with 4% paraformaldehyde for 30 minutes, permeabilized with 0.04% Triton X–100 in PBS, blocked in DMEM with 10% FBS. Primary antibodies were incubated at 4oC overnight and after washed 3 times with PBS, the secondary antibodies were incubated at 37oC for an hour. The nuclei were dyed with 4, 6-diamidino–2-phenylindole (DAPI) (Bioworld Technology, St Louis, USA). The cells were observed under a fluo rescence microscope at ×400 magnification and photographed.
Real-time PCR (RT-PCR)
RT-PCR was performed on an Applied Biosystems 7500 Real-time PCR System using SYBR Premix Ex Taq Kit (TaKaRa, Dalian, China) following the manufacturer’s protocols. Primers were obtained from Shanghai Sangon Biological Engineering Technology and Services (Shanghai, China) and their sequences were: PDCD4, forward primer 5′- CAG TTG GTG GGC CAG TTT ATT G –3′, reverse primer 5′- AGA AGC ACG GTA GCC TTA TCC A –3′; E-cadherin, forward primer 5′-ATG AGG TCG GTG CCC GTA TT–3′, reverse primer 5′- CGT TGG TCT TGG GGT CTG TGA–3′; N-cadherin, forward primer 5′-TCA GTG GCG GAG ATC CTA C–3′, reverse primer 5′-GTG CTG AAT TCC CTT GGC TA–3′; Vimentin, forward primer 5′-AAG CAG GAG TCA AAC GAG TA–3′, reverse primer 5′-GTT GGC AGA GGC AGA GAA AT–3′; GAPDH, forward primer 5′-CTC AAC TAC ATG GTC TAC ATG TTC CA–3′, reverse primer 5′-CTT CCC ATT CTC AGC CTT GAC T–3′. The level of GAPDH mRNA transcript was used to normalize all reported gene expression levels, and the data were analyzed using the 2-∆∆Ct method.
Animal studies
BALB/c nude mice were obtained from the Experimental Animal Center (License No. scxk (WAN) 2011–002) of Anhui Medical University (Anhui, China).Twenty nude mice were randomly divided into four groups. Stable PDCD4 high-expressed SGC–7901 cells were screened and confirmed. Mice were observed daily for tumor occurrence and growth for 30 days. The tumor dimensions and body weight of each mouse were measured every 3 days after treatment. On completion of the study, the mice were killed by cervical dislocation, and their tumors were excised and weighed.
Statistical analysis
All data were presented as Mean ± SD and performed using SPSS 20.0 statistical software. Statistical comparisons of more than two groups were performed using one-way analysis of variance (ANOVA) with Bonferroni’s post hoc test. P values below 0.05 were considered statistically significant.