All cell lines were authenticated by short-tandem repeat analysis. The human embryonic kidney cell line HEK293A (obtained in November 2009, authenticated in June 2015) and GC cell lines MKN45, SGC7901, MGC803, AGS, and HGC27 (obtained in July 2011, authenticated in June 2015) were obtained from the Committee of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). All cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified chamber containing 5% CO2.
Patients and tissue samples
The medical records of 172 GC patients treated at Sun Yat-sen University Cancer Center (Guangzhou, China) between January 2003 and December 2005 were reviewed. The patient selection criteria were as follows: (1) the patient was pathologically diagnosed with gastric adenocarcinoma; (2) the patient had received gastrectomy with limited or extended lymphadenectomy; (3) the patient did not receive any anticancer treatment before surgery; (4) the patient had complete clinical information, including follow-up data; (5) the patient had no other synchronous malignancies or familial malignancy; (6) the patient had no recurrent or remnant GC; and (7) the patient survived at least 3 months after surgery. Follow-up data were obtained through on-site interview, telephone calling or medical chart review. Overall survival (OS) was defined as the time from surgery to death from any cause or last follow-up. The study was approved by the Ethics Committee of Sun Yat-sen University Cancer Center (Guangzhou, China), and written informed consent was obtained from all participants.
Recombinant adenoviral expression vector construction and transfection
The TES recombinant adenoviral expression vector (Ad-TES) and control vector (Ad-Control) were constructed using the Gateway cloning system (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s protocol. After linearization by PacI enzyme, Ad-TES and Ad-Control were transfected into HEK293A cells using Lipofectamine 2000 (Invitrogen). After 10–13 days, when an approximately 80% cytopathic effect was observed, cells and medium were collected. After lysing the cells by three freeze–thaw cycles, the adenoviral supernatant was harvested by centrifugation (1000×g) at 4 °C for 15 min, tittered using Adenovirus Titer Immunoassay Kit (Innogent, Shenzhen, Guangdong, China) and stored at − 80 °C. To increase the transfection efficiency, HEK293A cells were re-infected with the initial harvested viral supernatant. Three to 4 days later, the cell lysates were collected after three freeze–thaw cycles. MKN45 or SGC7901 cells were transfected with Ad-TES at a multiplicity of infection (MOI) of 200. The transfection efficiency was calculated by dividing the amount of cells presenting green fluorescence by the total number of attached cells in 10 fields randomly selected for each sample under a fluorescence microscope with 100× magnification.
Extraction of total RNA and reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was extracted using TRIzol (Invitrogen) according to the manufacturer’s protocol. The concentration of total RNA was assessed by measuring absorbance at 260 nm using a NANO DROP spectrophotometer (ND-1000, Thermo Scientific, Waltham, MA, USA). Two mg of total RNA was reversely transcribed into cDNA using M-MLV reverse transcriptase (Promega, Madison, Wisconsin, USA) according to the manufacturer’s recommendation. The cDNA templates were amplified using the specific primer set for TES. The samples amplified with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primer set were used as an internal control. Primers used in this study were as follows: the forward primer 5′-CATGGACCTGGAAAACAAAGTG-3′ and the reverse primer 5′-CTAAGACATCCTCTTCTTACATTCCAC-3′ for TES; the forward primer 5′-CGGGAAGCTTGTCATCAATGG-3′ and the reverse primer 5′-G GCAGTGATGGCATGGACTG-3′ for GAPDH. The corresponding PCR products were 1267 bp for TES and 358 bp for GAPDH.
Protein extraction and western blotting
Forty-eight h after adenoviral transfection, protein expression was examined by Western blotting. GC cells (MKN45, SGC7901, MGC803, AGS, and HGC27) were lysed in RIPA lysis buffer (Beyotime, Shanghai, China), and lysates were harvested by centrifugation (13,000×g at 4 °C for 30 min. Western blotting was carried out as we previously described , using GAPDH as an internal control. The following primary antibodies and secondary antibodies were used:
A mouse monoclonal antibody against TES (1:500 dilution; Santa Cruz, Dallas, TX, USA), a rabbit monoclonal antibody against Mena (1:1000 dilution; Cell Signaling Technology, Boston, MA, USA), a rabbit polyclonal antibody against Lpd (1:1000 dilution; Sigma, St.Louis, MI, USA), HRP-conjugated rabbit anti-mouse IgG antibody (1:2000 dilution; Santa Cruz) and HRP-conjugated goat anti-rabbit IgG antibody (1:2000 dilution; Epitomics, Burlingame, CA, USA), a HRP-conjugated mouse anti-human GAPDH monoclonal antibody (1:5000 dilution; Shanghai Kangchen, Shanghai, China).
MKN45 or SGC7901 cells were seeded in RPMI-1640 medium supplemented with 10% FBS in 96-well plates (500 cells per well). CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega, Beijing, China) was used to assess cell viability according to the manufacturer’s instruction for 5 consecutive days. Triplicate independent experiments were performed. The proliferation rate was calculated as follows: proliferation rate = (ODn− OD1)/OD1 × 100% (n: days after transfection).
Colony formation assay
MKN45 or SGC7901 cells were seeded onto 6-well plates (500 cells per well). After cultured in RPMI-1640 medium supplemented with 10% FBS for 12 days, surviving colonies (> 50 cells per colony) were counted after 0.5% (m/v) crystal violet staining. Colony-forming efficiency (CFE %) was defined as the ratio of the number of colonies formed to the number of cells inoculated. Triplicate independent experiments were performed.
Cell cycle analysis
Forty-eight h after adenoviral transfection, GC cells were collected, washed twice with precooled phosphate buffer sodium (PBS) and fixed with ice-cold 75% ethanol at − 20 °C for 1 h. After washing with PBS, cells were resuspended in ice-cold PBS (500 µL) containing RNAase (100 µg) and incubated at 37 °C for 30 min, followed by propidium iodide (PI) staining at 4 °C for 30–60 min. Cell cycles of MKN45 and SGC7901 cells were analyzed on a FC500 flow cytometer (Beckman, Brea, CA, USA) using the Cylchred software (University Wales College Medicine, Cardiff, UK).
Cell invasion and migration assay
Cell invasion and migration assay were performed using MKN45 and SGC7901 cells transfected with Ad-TES or Ad-Control. The invasion assay was performed in a Transwell comprising a polycarbonate membrane with 8-μm pores (Corning, Shanghai, China) placed in a 24-well plate. The Transwell insert was coated with 50 µL Matrigel Basement Membrane Matrix (BD Biosciences, Bedford, MA, USA). Cells in 100 μL of RPMI-1640 medium without FBS were added to the Transwell insert, and 0.5 mL of RPMI-1640 medium containing 20% FBS was placed in the lower chamber. The cells were incubated at 37 °C and allowed to invade through the Matrigel layer. After 48 h, cells on the lower surface of the Transwell insert were fixed with 75% methanol and stained with 0.5% (m/v) crystal violet. The stained cells were counted in 10 random fields with 200× magnification. The migration assay was similar to the invasion assay, except that the Transwell insert was uncoated. Each experiment was performed in triplicate.
MKN45 and SGC7901 cells were washed twice with ice-cold PBS and resuspended in 400 µL 1× Binding Buffer (Bestbio, Shanghai, China), according to the manufacturer’s protocol. After incubation with 5 µL Annexin V-FITC (Bestbio) for 15 min at room temperature in the dark, 10 µL PI (Bestbio) was added, and the cells stained with PI and Annexin V were counted using flow cytometer (Beckman).
Small interfering RNA (siRNA) transfection
siRNA targeting Mena (siMena, synthesized by GenePharma Company, Shanghai, China) were used to knockdown Mena expression in MKN45 and SGC7901 cells. The cells were seeded in 6-well plates (1 × 106 cells per well). When an approximately 50% cell confluence was observed, the cells were transfected with 400 pmol of siMena or siNC (negative control) at 37 °C for 48 h using Lipofectamine RNAi MAX reagent (Invitrogen), according to the manufacturer’s instructions. The expression level of Mena was examined 48 h after the transfection. siRNA sequences used in this study were as follows:
Sense sequence 5′-GGUCCUAUGAUUCAUUACATT-3′ and antisense sequence 5′-UGUAAUGAAUCAUAGGACCTT-3′ for siMena 1;
Sense sequence: 5′-GCGAGAAAGAAUGGAAAGATT-3′ and antisense sequence 5′-UCUUUCCAUUCUUUCUCGCTT-3′ for siMena 2;
Sense sequence: 5′-UUCUCCGAACGUGUCACGUTT-3′ and antisense sequence 5′-ACGUGACACGUUCGGAGAATT-3′ for siNC.
Immunoprecipitation-based mass spectrometry (IP-based MS)
A Pierce® Classic IP Kit (Thermo Scientific) was used to perform the protein interaction assay, according to the manufacturer’s protocol. In brief, after lysing in IP Lysis/Wash Buffer (0.025 mol/L Tris, 0.15 mol/L NaCl, 0.001 mol/L EDTA, 1% NP-40, and 1% protease inhibitor cocktail) at 4 °C for 5 min, cell lysates were collected by centrifugation (13,000×g) at 4 °C for 10 min. The supernatants were transferred to new tubes immediately. Total proteins were quantified with BCA Protein Quantification Kit (Beyotime) and adjusted to 3.5 μg/μL with PBS. An amout of 10 μg primary antibody against the target protein were added and incubated at 4 °C overnight. The mixtures were then incubated with 20 μL Protein A/G Plus Agarose for 6 h at 4 °C. After incubation, the Protein A/G Plus Agarose were washed three times with IP Lysis/Wash Buffer. Collected precipitates were eluted with 50 μL elution buffer at 100 °C for 10 min. Eluted proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the protein bands were stained with silver solution (1×; Beyotime). The bands of interest were analyzed using MS (Institutes of Life and Health Engineering, Jinan University, Guangzhou, Guangdong, China). The antibodies used in immunoprecipitation were as follows: a mouse monoclonal antibody against TES (1:100 dilution; Sigma) and a rabbit monoclonal antibody against Mena (1:50 dilution; Cell Signaling Technology).
Tumorigenicity assays in nude mice
Five-week-old BALB/c nude mice (Guangdong Medical Experimental Animal Center, Guangzhou, Guangdong, China) were randomly assigned to four groups (7 mice per group) before inoculation. At 48 h after adenoviral transfection, MKN45 and SGC7901 cells were injected subcutaneously into the groin of mice respectively (5 × 106 cells suspended in 200 μL PBS per mouse). The length (L) and width (W) of the tumor was measured with calipers every 3 days. The tumor volume was calculated as (L × W2)/2. Thirty-two days after tumor inoculation, the mice were sacrificed by cervical dislocation, and tumor weight was assessed.
Metastasis assay in nude mice
To investigate the suppressive effect of TES on tumor metastasis, 1 × 106 cells (MKN45 cells transfected with Ad-TES or Ad-Control) in 200 μL PBS were injected intravenously through the lateral tail vein into 5-week-old nude mice (8 mice per group). After 6 weeks, the mice were necropsied after anesthesia. Their lungs were fixed in 3.7% formaldehyde, 5% glacial acetic acid, and 72% ethanol for 24 h before proceeding to paraffin embedding. Serial 5-μm sections were stained with hematoxylin and eosin (H&E) for histopathological examination. Metastasis lesions from 10 random high-power fields were counted. All animal experiments were conducted in compliance with the guidelines of the laboratory animal ethics committee of Sun Yat-sen University (Guangzhou, Guangdong, China).
Immunohistochemistry (IHC) and semi-quantitative analysis
IHC was performed on GC tissue sections as previously described . The antibodies used in IHC were as follows: a mouse monoclonal antibody against TES (1:300 dilution; Santa Cruz) and a rabbit monoclonal antibody against Mena (1:500 dilution, Cell Signaling Technology). The intensity and extent of immunostaining were evaluated for all tumor samples by three pathologists under double-blinded conditions. In brief, the percentage of positive staining was scored as 0 (0–9%), 1 (10–25%), 2 (26–50%), or 3 (51–100%), and the intensity as 0 (no staining), 1 (weak staining), 2 (moderate staining) or 3 (dark staining). The total immunostaining score was calculated as the product of extent and intensity, ranging from 0 to 9.
Based on IHC scores, expression levels of Mena and TES were defined as low (score 0–3) or high (score 4–9) in subgroups.
Student t test was performed for comparison of continuous variables between groups. Repeated measurement analysis of variance (ANOVA) analysis was used to compare curves of tumor growth. Chi square test was used to examine differences of categorical variables between subgroups. Kaplan–Meier survival curve was plotted and compared by log-rank test. The patients did not have an event during the observation time were described as censored. Multivariate Cox proportional hazards regression model was used to calculate the hazard ratio (HR) and 95% confidence interval (CI) for prognosis evaluation. All statistical analyses were performed using the SPSS Statistics 22.0 software (IBM, Armonk, NY, United States), and P < 0.05 was considered statistically significant.