Down-regulation of WAVE2, WASP Family Verprolin-Homologous Protein 2, in Gastric Cancer Indicates Lymph Node Metastasis and Cell Migration

Materials and Methods

Chemicals. We purchased the following materials from Life Technologies (Paisley, Scotland, UK): Polymerase chain reation (PCR) primers, molecular biology grade agarose, DNA ladder, pEF6/V5-His-TOPO plasmid vector and competent One Shot™ TOP10 Esherischia coli. TRI Reagent was from Sigma (Sigma-Aldrich, Inc., Poole, Dorset, UK) and first-strand cDNA was synthesized using iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Mastermix for routine PCR and quantitative PCR were from Thermo Fisher Scientific (Surrey, England, UK). WAVE2 and ARP2 antibodies were purchased from Santa Cruz Inc. (Santa Cruz, CA, USA) (cat. number: sc-33548 and sc-137250). The ARP2/3 inhibitor, 8102-5105 (cat. number: 182515), and NWASP inhibitor, wiskostatin (cat. number: 681525), were from Calbiochem (Nottingham, England, UK). ROCK inhibitor, Y-27632 was from Santa Cruz Biotechnology (cat. number: sc-3536). Matrigel was from BD Bio-Science (cat. number: 354234) (Oxford, England, UK). ECIS 96W1E+ electrical arrays were from Applied Biophysics Inc. (ECIS™, NY, USA).

Human gastric cancer tissues, slides and cells. A total of 324 patients (male: 231 cases, female: 93 cases; mean age=59.8 years; range=23-87 years, median survival=24 months) with gastric cancer, who were diagnosed and surgically treated at Peking University Cancer Hospital between 2004 and 2007, were enrolled into this study. Corresponding slides were collected from the archived sections. The study was approved by the local Ethics Committee (Ethics Number: 2006021) and consent was obtained from patients. Some of the patients had received chemotherapy or radiation therapy preoperatively. The following histopathological information was recorded: the depth of tumour invasion, histological grade, and status of lymph node metastasis and liver metastasis. Stage of gastric adenocarcinoma was classified according to 1997 tumour-node-metastasis (TNM) classification recommended by the International Union Against Cancer. All patients were followed up until June 2012. Gastric cell line HGC27 was acquired from the European Collection of Animal Cell Culture (ECACC, Salisbury, UK). Cells were maintained in DMEM-F12 medium supplemented with 10% foetal bovine serum (FBS) and antibiotics.

RNA preparation, RT-PCR and quantitative analysis of transcripts of WAVE2 and Epithelial-Mesenchymal Transition (EMT) markers. Frozen tissues or cells from a 25 cm² culture flask (50-100 mg) were homogenized and placed into 1 ml of TRI reagent for RNA extraction. The homogenization was then precipitated with chloroform and isopropanol. The RNA pellet was resuspended in DEPC treated water. The concentration of the RNA was determined using a UV spectrophotometer. First-strand cDNA was synthesized using iScript cDNA Synthesis Kit, and the quality of the cDNA was verified using GAPDH primers. Conventional PCR was performed using GreenTaq ReadyMix PCR reaction mix. Cycling conditions were 94°C for 5 minutes, followed by 28 to 30 cycles at 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 40 seconds. This was followed by a final extension of 10 minutes at 72°C. The products were visualized in 1% agarose gels stained with SYBR Green. Q-PCR was performed using IcyclerIQ™ (Bio-Rad, Hemel Hempstead, UK), based on the Amplifluor™ technology (14) modified from a previously reported method (15). Cytokeratin-19 (CK19) was also run in parallel during the 96 cycles of analysis to enable normalization of data. The reaction was carried out under the following conditions: 94°C for 5 minutes, 96 cycles of: 94°C for 15 seconds, 55°C for 35 seconds and 72°C for 20 seconds. The levels of the transcripts were generated using an internal standard that was simultaneously amplified with the samples, and are shown here in two ways: based on equal amounts of RNA, and as a target:CK19 ratio (16). All the primers used are listed in Table I.

Immunohistochemical staining of WAVE2 and ARP2 proteins. Immunohistochemistry was performed in 127 cases of pathological sections. In order to clarify the localization of WAVE2 and ARP2, we prepared 40 cases of serial mirror sections from a pair of consecutive specimens. These were placed on slides with the common cross-section turned upwards, so that they would share the same cutting surface as described by Iwava et al. (17). Briefly, 4-μm sections from formalin-fixed, paraffin-embedded tissues were mounted on poly-L-lysine-coated glass slides and then de-paraffinized in xylene and rehydrated through alcohol to distilled water. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 15 min at room temperature. After pressure cooking the sections in 10 mmol/l EDTA (pH 8.0) for 3 min, the sections were incubated at room temperature with antibody to WAVE2 (1:100 diluted) or ARP2 (1:200 diluted) for 1 h, followed by horse-raddish peroxidase (HRP)-conjugated secondary antibody. Development of slides was performed using peroxidase substrate (diaminobenzidine tetrahydrochloride) solution, followed by counterstaining with haematoxylin, dehydration in ethanol, and clearing with xylene. GC tissues treated with PBS (phosphate buffered saline) as primary antibody was used as negative control for both. The degree of WAVE2 and ARP2 antibody reactivity was scored by assessing the percentage of stained adenocarcinoma cells in the section. In this study, adenocarcinoma specimens and background normal tissues were defined as ‘negative’ (scored 0, for negative and 10% or fewer stained cells) and ‘positive’ expression (scored 1, for more than 10% positively stained cells). Each pair of mirror section was compared carefully to determine whether ARP2 and WAVE2 staining revealed the same localization and the same intracytoplasmic distribution within the tumour and normal cells. If more than 5% of the cells expressed both WAVE2 and ARP2 in their cytoplasm, they were judged to be positive for coexpression (17). Slides were analyzed independently by two observers using light microscopy. Photographs were recorded on an Olympus CKX41 microscope (Bristol, England, UK).

Construction of anti-WAVE2 hammerhead ribozyme transgenes. Anti-human WAVE2 hammerhead ribozymes were designed to knockdown the WAVE2 gene based on the secondary structure of the mRNA generated using Zuker's RNA Mfold programme (18). The ribozymes were synthesized and cloned into pEF6/V5-His-TOPO plasmid vector and then ribozyme transgenes and control plasmids were then transfected into HGC27 cells using an electroporator (Easject Plus; EquiBio Ltd., Kent, UK). Transient transfectants were obtained and verified after 36 hours. RT-PCR was used to verify the efficiency of transient transfection.

In vitro cell growth assay. This was based on a procedure previously described (19). GC cells were plated onto a 96-well plate (3,000 cells /well). Cells were fixed in 4% formalin after 1, 3, and 5 days. The cells were then stained with 0.5% (w/v) crystal violet for 30 minutes. Following washing, the crystal violet stain was extracted with 10% (v/v) acetic acid. Absorbance was determined at a wavelength of 540 nm using a spectrophotometer (Elx800; Bio-Tek, Bedfordshire, UK). Growth rate was calculated as: growth rate at day 5 (%)=(absorbance at day 5/absorbance at day 1) ×100.

In vitro invasion assay. This was carried out as previously reported and modified in our laboratory (20). Transwell inserts with 8 μm pore size were coated with 50 μg matrigel and air-dried. The matrigel was rehydrated before use. A total of 25,000 cells were seeded into each well and after 72 hours, cells were fixed (4% formalin), stained with crystal violet, and counted.

Electric cell-substrate impedance sensing (ECIS)-based cell adhesion and motility assays. ECIS is a novel method used as an alternative to the conventional function assays. It works with an array of 96 wells, each containing two gold electrodes. The current and voltage across this electrode are measured, and the impedance and resistance calculated. From the impedance changes, effects on cell attachment and motility can be examined (21). Using the ECIS 96W1E+ array, cell adhesion and wounding assay were also conducted. A total of 40,000 cells diluted in 100 μl DMEM with 20% Foetal Calf Serum were seeded into each ECIS plate well, and treated with a protein of interest. In this study, cells were treated with either ARP2/3 inhibitor, 8012-5102; NWASP inhibitor, wiskostatin; or ROCK inhibitor, Y-27632 at a final concentration of 200 nM. For the control group, an identical volume of serum-free medium was added to wells. The array was then placed into an ECIS™ CO₂ incubator which was connected to the ECIS™ Model 9600 Controller. Cell adhesion was assessed within the first 40 minutes and electrical wound was set at the 16th hour when the resistance reached maximum levels and migration data could be obtained in the subsequent 6 hours.

Statistical analysis. Statistical analysis was performed using SPSS18 (SPSS Inc., Chicago, IL, USA). Q-PCR data were analyzed using Mann–Whitney U-test for non-normally distributed data. The association of staining for WAVE2 in GC with patient survival was evaluated using life tables constructed from survival data with Kaplan–Meier plots and analysed using log-rank statistics. Overall survival was measured from date of initial surgery to date of death, counting death from any cause as the endpoint, or the last date of information as the endpoint if no event was documented. The association of the transcript levels of WAVE2 and EMT markers was analysed using Spearman rank order correlation analysis. Other normally distributed data were analyzed using Student's t-test. Each assay was performed at least three times. p-Values of less than 0.05 were considered statistically significant.