Cell culture and tissue collection
The human GC cell lines SGC7901, BGC823, MKN45, and AGS, and the immortalized gastric epithelial cell line GES were purchased from the Cell Resource Center of the Chinese Academy of Sciences, Shanghai, China. Cells were maintained in Dulbecco’s Modified Eagle’s Medium (Thermo Scientific HyClone, Beijing, China) supplemented with 10% fetal bovine serum (HyClone), 100 U/mL penicillin, and 100 U/mL streptomycin (HyClone) in a 37 °C humidified incubator with a mixture of 95% air and 5% CO2.
A total of 20 fresh primary GC samples and matched adjacent non-cancerous tissues were obtained from patients undergoing surgery at Xijing Hospital, Xi’an, China. All samples were confirmed by the Department of Pathology at Xijing Hospital and stored in a liquid nitrogen canister. All patients provided informed consent for excess specimens to be used for research purposes and all protocols employed in the present study were approved by the Medical Ethics Committee of Xijing Hospital.
Mice
Female BALB/c nude mice were provided by the Experimental Animal Center of the Fourth Military Medical University and were housed in pathogen-free conditions. All animal studies complied with the Fourth Military Medical University animal use guidelines, and the protocol was approved by the Fourth Military Medical University Animal Care Committee.
Reagent and inhibitor
5-Fluorouracil was purchased from Sigma (Sigma-Aldrich Corporation, Los Angeles, CA, USA), and MAPK/ERK inhibitor (PD98059) and p53 inhibitor (A15201) were purchased from Invitrogen (Thermo Fisher Scientific, Cambridge, Massachusetts, USA); all were stored according to the manufacturer’s instructions.
RNA extraction and real-time polymerase chain reaction (PCR)
Total RNA was extracted from cell lines using the RNeasy Plus Universal Tissue Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The PCR primers for DDIT4 and ACTB were synthesized by TaKaRa (Dalian, China). The sequences were as follows: DDIT4, 5′-GGACCAAGTGTGTTTGTTGTTTG-3′ (Forward) and 5′-CACCCACCCCTT CCTACTCTT-3′ (Reverse); ACTB, 5′-TCATGAAGTGTGACGTTGACATCCGT- 3′ (Forward) and 5′-CCTAGAAGCATTTGCGGTGCACGATG-3′ (Reverse). cDNA was synthesized using the PrimeScript RT Reagent Kit (TaKaRa, Dalian, China). Real-time PCR was performed using SYBR Premix Ex Taq II (TaKaRa). Fluorescence was measured using a LightCycler 480 system (Roche, Basel, Switzerland). ACTB was used as an internal control for mRNA analysis. Each sample was run in triplicate.
Protein extraction and western blotting
Total proteins were prepared from fresh frozen tissue or cultured cells in radio immunoprecipitation assay (RIPA) lysis and extraction buffer (Beyotime Biotechnology, Shanghai, China) with protease and phosphatase inhibitors. Denatured proteins (20–50 mg) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The following primary antibodies were used according to the manufacturer’s instructions: anti-DDIT4 (Dilution 1:500, Abcam, Cambridge, MA, USA) and anti-β-actin (Dilution 1:2000), anti-Ki67 (Dilution 1:1000), anti-p53 (Dilution 1:1000), anti-p-p53 (p-Ser6) (Dilution 1:1000), anti-p-p53 (p-Ser315) (Dilution 1:1000), anti-p21Cip1 (Dilution 1:500), anti-p-p21Cip1 (p-Thr145) (Dilution 1:500), anti-MEK1 (Dilution 1:1000), anti-p-MEK1 (p-Ser221) (Dilution 1:1000), anti-p42/44MAPK (Dilution 1:1000), and anti-p-p42/44MAPK (p-Thr202 and p-Tyr204) (Dilution 1:1000) (Cell Signaling Technology, Beverly, MA, USA). Densitometry of specific blotted bands was analyzed by ImageJ 1.48 software (Image-Processing and Analysis in Java; National Institutes of Health, Bethesda, MD, USA; http://imagej.nih.gov/), and the intensity values were normalized against the β-actin loading control.
Tissue microarray immunohistochemistry
GC tissue microarrays were purchased from Superchip (Shanghai, China). Each array contained 90 cases of paired adjacent gastric tissues and primary GC tissues. Immunohistochemical (IHC) staining was performed using anti-DDIT4 and anti-Ki67 antibodies as per the manufacturer’s instructions. IHC results were scored independently by two pathologists in a blinded manner. The scoring was based on the intensity and extent of staining. Staining intensity was graded as follows: 0, negative staining; 1, weak staining; 2, moderate staining; and 3, strong staining. The proportion of stained cells per specimen was determined semi-quantitatively as follows: 0, < 1%; 1, 1–25%; 2, 26–50%; 3, 51–75%; and 4, > 75%. The histological score (H-score) for each specimen was computed using the following formula: H-score = proportion score×intensity score. The samples were further characterized by H-score as negative (−, score: 0), weak (+, score: 1–4), moderate (++, score: 5–8), and strong (+++, score: 9–12). Samples with an H-score of > 4 were considered to exhibit high expression, and samples with an H-score of ≤ 4 were considered to exhibit low expression.
Immunofluorescence staining
Cells were plated onto glass coverslips, fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 in PBS for 15 min. Blocking solution was applied at room temperature for 1 h. Rabbit anti-human DDIT4 primary antibody (Abcam) was applied at 4 °C overnight. FITC-conjugated goat anti-rabbit and Cy3-conjugated goat anti-mouse secondary antibodies were incubated on the coverslips at room temperature for 2 h. Immunostaining signals and DAPI-stained nuclei were visualized at room temperature using a confocal microscope (FV10i; Olympus, Tokyo, Japan) equipped with a 10×/0.30 numerical aperture objective lens (Olympus) and Fluoview software (version 4.3; Olympus). No imaging medium was used. For better visualization, the images were adjusted using the level and brightness/contrast tools in Photoshop according to the guidelines for the presentation of digital data.
Lentivirus infection
DDIT4-overexpression or sh-DDIT4 lentivirus infection was conducted by GeneChem (Shanghai, China). Target cells (1 × 105) were infected with 1 × 107 lentivirus-transducing units in the presence of 5 mg/mL polybrene. An empty lentiviral vector was used as a negative control. After transfection and antibiotic selection for 6 weeks, cells were collected for further investigation.
High-content screening assay
Cells transfected with lentivirus stably expressing green fluorescent protein (GFP) were seeded into 96-well plates and treated with increasing doses of 5-FU (0, 10, 20 µg/mL for GC cells or 0, 2, 4 µg/mL for GES cells). Cells were imaged every 4 h for 48 h using the Thermo Scientific CellInsight CX7 High Content Analysis Platform (0, 10, 20 µg/mL for GC cells or 0, 2, 4 µg/mL for GES cells; Thermo Fisher Scientific, Cambridge, Massachusetts, USA). Proliferation curves were plotted and analyzed using HCS Studio Software (Thermo Fisher Scientific).
Colony formation assays
Transfected cells were seeded in six-well plates (1000 cells/well). After 14–18 days of incubation to establish stable clones, cells were fixed with 70% ethanol and stained with crystal violet solution. Colonies containing greater than 50 cells were counted. The experiment was conducted with three independent triplicates.
Cell cycle and apoptosis assays
For cell cycle analysis, target cells were selected with antibiotics (penicillin–streptomycin solution) for 48 h after transfection as indicated, fixed in 75% ethanol, and stained with propidium iodide supplemented with RNase A (Roche, Mannheim, Germany) for 30 min at 22 °C. The Annexin V-FITC Apoptosis Detection Kit (Cell Signaling Technology, Beverly, MA, USA) was used for apoptosis assays. Cells (1 × 104) were stained according to the manufacturer’s protocol and sorted using a fluorescence-activated cell sorting sorter (BD Biosciences, La Jolla, CA, USA). Data were analyzed using ModFit software (BD Biosciences).
In vivo tumorigenicity
BGC823 cells (5 × 105 cells in 0.2 mL of PBS) transfected with pCMV-DDIT4 or empty pCMV were injected subcutaneously into the dorsal flank of 5-week-old female Balb/c nude mice (five mice per group). Tumor diameter was measured every 3 days for 30 days. Tumor volume (mm3) was calculated based on the longest and shortest diameters as follows: volume = (shortest diameter)2 × (longest diameter) × 0.5. Thirty days after injection, all mice were killed, and the tumor xenografts were isolated for further analysis. All experimental animals were supplied by the Experimental Animal Center of the Fourth Military Medical University. All protocols for animal studies were approved by the Fourth Military Medical University Animal Care Committee.
Cell counting kit-8 (CCK8) assay
For cell counting kit 8 assays, cells were seeded into 96-well plates at a density of 1000 cells in 100 μL of complete culture medium per well. At the indicated time points, the medium was replaced with a kit solution (TransDetect cell counting kit, Transgene, Beijing, China) and complete culture medium at a ratio of 1:9, and the samples were incubated for 2 h at 37 °C. The absorbance of each sample was analyzed at 450 nm using a microtiter plate reader (Tecan, Switzerland). The assay was repeated in triplicate.
Phospho-specific protein microarray analysis
Phospho-array detection was performed in cooperation with Wayen Biotechnology (Shanghai, China). At 48 h post-transfection, all treated cells were collected for protein extraction. Protein samples of 50 mg each were tagged with biotin reagent and hybridized on a Phosphorylation ProArray (Full Moon BioSystems, USA) using an Antibody Array Kit (FullMoon BioSystems, USA) for the detection of 248 site-specific cancer signaling phospho-antibody profiles. Finally, fluorescence intensity was scanned by GenePix 4000B (Axon Instruments, Houston, USA) using GenePix Pro 6.0. The raw data were manipulated using Grubbs’ method. The phosphorylation ratio was calculated as follows: phosphorylation ratio ¼ phospho value/unphospho value.
Statistical analyses
SPSS software (version 19.0, SPSS Inc., Chicago, IL, USA) was used for statistical analyses. Continuous data are presented as the mean ± standard deviation (SD), and Student’s unpaired t-test was utilized for comparisons between two groups. Frequencies of categorical variables were compared using the χ2 test. A P value of less than 0.05 was considered significant.