XB130 promotes proliferation and invasion of gastric cancer cells

Several common human gastric adenocarcinoma cell lines [(BGC823), (SGC7901), (MKN45), (MKN28), (AGS)] were obtained from Foleibao Biotechnology Development Company (Shanghai, China). Cells were cultured in complete medium [Roswell Park Memorial Institute 1640 medium (Life Technologies, Carlsbad, CA, USA) with 10% fetal bovine serum (Thermo Scientific HyClone, South Logan, UT, USA)] at 37°C under 5% CO₂. Cells were harvested in the logarithmic growth phase for use in the experiments described below. Silencing of XB130 was carried out using small hairpin RNA (shRNA) as described previously [4]. The sequences were GCTGAAGATCACACCGATG for XB130-silencing shRNA and GCCAGCTTAGCACTGACTC for Scramble shRNA, respectively. Establishment of cell lines transfected with XB130 shRNA (sh-XB130) was performed as described previously [4].

Rabbit antibodies for fibronectin and CD44, as well as mouse antibodies for E-cadherin, vimentin, α-catenin, β-catenin, XB130 and β-actin were purchased from Santa Cruz Biotechnology Company (Santa Cruz, CA, USA). Rabbit antibodies for β-actin, Akt, and p-Akt were purchased from Cell Signaling Technology Company (Boston, MA, USA), while a rabbit antibody targeting XB130 was obtained from PradoWalnut Company (Walnut, CA, USA).

Primer sequences for human XB130 were (F) 5′-AAGCAGCAGCTCTGATGAGG-3′ and (R) 5′-GGTCTGGAAGGCTCTTCTGA-3′. Total RNA was extracted from cultured cells using a Trizol kit (Life Technologies, Carlsbad, CA, USA). Then cDNA was synthesized using total RNA and MMLV-RT reverse transcriptase (ProSpec, East Brunswick, NJ, USA). The reaction mixture for RT-PCR was prepared according to the manufacturer’s protocol.

Cells were lysed on ice in RIPA buffer [50 mM Tris-Cl (pH 7.5), 120 mM NaCl, 10 mM NaF, 10 mM sodium pyrophosphate, 2 mM EDTA, 1 mM Na₃VO₄, 1 mM PMSF, and 1% NP-40] containing a protease inhibitor cocktail (Roche, Basel, CH). The protein content of the lysates was determined by the method of Bradford. Approximately 50–75 μg of protein was resolved by 8% or 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and was transferred to nitrocellulose membranes (Millipore, Bedford, MA, USA). The membranes were blocked in TBST [25 mM Tris–HCl (pH 7.5), 125 mM NaCl, and 0.1% Tween 20] containing 5% bovine serum albumin (BSA), and then incubated with primary antibodies targeting XB130, E-cadherin, α-catenin, β-catenin, fibronectin, MMP9, MMP2, vimentin, CD44, Akt, p-Akt, or β-actin in TBST containing 1% BSA overnight at 4°C. Subsequently, incubation was done with the appropriate secondary antibodies for 1 h at room temperature. Reactive protein bands were visualized with a Western Lightning Plus-ECL (Perkin Elmer, Waltham, MA, USA) after exposure to radiographic film and were quantified with QuantityOne v4.6.2 imaging software (Bio-Rad, Hercules, CA, USA).

To investigate the ability of cells to form colonies, 1×10³ cells transfected with XB130 shRNA or Scramble RNA were seeded into 6-well plates and incubated for 2 weeks with a medium change every 3–4 days. Colonies were stained with 0.05% crystal violet (Sigma Chemical Company, Louis, MO, USA) for 1 h at room temperature, washed twice with phosphate-buffered saline (PBS), and observed under a microscope (Olympus, Tokyo, Japan).

Cells were trypsinized and suspended in 2 mL of complete medium with 0.3% agar (Sigma Chemical Company, Louis, MO, USA), and then the agar-cell mixture was plated onto the bottom layer with 1% agar in complete medium. After being cultured in an incubator for 4 weeks, cells were observed and photographed under a microscope.

After trypsinization, cells were seeded into 96-well plates at a density of 0.2×10⁴/well for culture, and cell proliferation was measured by the methyl thiazolyl tetrazolium (MTT) assay on days 1, 3, 5, and 7. Briefly, 0.02 mL of MTT solution (5 mg/mL in PBS) was added to each well and incubation was performed for 4 h at 37°C, after which the medium was replaced by 0.15 mL of dimethyl sulfoxide and incubation was done for 10 min. Then the optical density was measured at 492 nm with a Microplate spectrophotometer (Thermo Scientific, Pittsburgh, PA, USA).

Cell cycle analysis was performed by flow cytometry (Beckman-coulter, Fullerton, CA, USA) after staining the cells with propidium iodide (PI) (Sigma Chemical Company, Louis, MO, USA). Cells were harvested by trypsinization, washed with PBS, and fixed in 70% ethanol for 30 min on ice. Then the cells were washed again, resuspended in PBS containing Triton-X-100 and 2 mg/mL RNase A (Thermo Scientific, Pittsburgh, PA, USA), and incubated at 37°C for 30 min. Next, PI was added at a final concentration of 25 μg/mL and the cells were incubated on ice for 30 min. After staining with PI had been completed, a minimum of 10,000 events were counted for each sample by flow cytometry and the cell cycle profile was analyzed with Flowjo software (TreeStar, Ashland, Oregon, USA).

The effect of XB130 inhibition on DNA synthesis was determined by estimating the uptake of 5-bromo-2′deoxyuridine-5′monophophate (BrdU) into DNA. Cells in the logarithmic growth phase were trypsinized, transferred to a sterile coverslip, and incubated until they became adherent. After serum starvation for 48 h and incubation in complete medium for 4 h, the cells were labeled with 10 μmol/L BrdU for 1 h. Then the cells were fixed and permeablilized with 0.1% Triton and 0.1% citric acid for 10 min at room temperature, after which endogenous peroxidase was blocked by incubation with 3% hydrogen peroxide (H₂O₂) for 10 min at room temperature. For nuclear staining, cells were incubated in serum-free medium with anti-BrdU antibody for 1 h at 37°C. Each experiment was repeated 3 times independently, and stained cells were counted under a fluorescence microscope (Olympus, Tokyo, Japan).

SGC7901 and MNK45 cells were seeded into 6-well plates at 90% confluence and incubated overnight for adherence. Then a wound was made along the center of each well by scratching the cell layer with the tip of a 200 μL pipette. Next, the wells were washed twice with PBS to remove loose cells and fresh medium was added. Photographs were taken at 0 h, 10 h, and 24 h to assess cell migration into the wound.

The invasive potential of wild-type and XB130-silenced GC cells was assessed by an invasion assay using 24-well Matrigel invasion chambers (BD Biosciences, San Jose, CA, USA). Briefly, Matrigel inserts and an equal number of control inserts were prepared according to the manufacturer’s protocol. SGC7901 cells and MNK45 cells (5×10⁴/mL in 0.5 mL of serum-free medium) were added to the upper chambers, and 0.75 mL of medium supplemented with 5% fetal bovine serum was added to each of the lower chambers as a chemoattractant. After incubation for 22 h, the cells remaining in the upper chambers were removed by scraping, and the invading cells in the lower chambers were fixed with 3.7% paraformaldehyde. Then the cells were washed twice with PBS, stained with hematoxylin for 1 h at room temperature, and photographed under a microscope.

Twenty-four–well dishes were coated with 100 μL of growth factor reduced solidified Matrigel (BD Biosciences, San Jose, CA, USA) and placed in an incubator. The cells were trypsinized and were seeded at a density of 500 per well in 500 μL of medium. After incubation for 2 weeks, the cultures were photographed under a microscope.

Cells were grown on coverslips, fixed with 4% paraformaldehyde for 30 min, and washed three times with PBS. Then the cells were permeabilized with 0.2% Triton X-100 for 5 min at room temperature and blocked with 1% BSA for 1 h. Next, incubation was done with primary antibodies targeting XB130, E-cadherin, and vimentin overnight at 4°C, followed by incubation with appropriate secondary antibodies (Alexa Fluor® 594 or Alexa Fluor® 488) for 1 h at room temperature. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (Life Technologies, Carlsbad, CA, USA), while F-actin filaments were stained with rhodamine phalloidin (Sigma Chemical Company, Louis, MO, USA), and the cells were viewed with a confocal laser-scanning microscope.

Six-week-old Balb/c nude mice were purchased from Sun Yat-Sen University (Guangzhou, China). All experimental procedures involving animals were done in accordance with the Guide for the Care and Use of Laboratory Animals and conformed to our institutional ethical guidelines for animal experiments. ShXB130-transfected, empty plasmid-transfected, and untransfected SGC7901 cells were trypsinized, collected by centrifugation, and suspended in RPMI-1640 medium. Then 0.2 mL of medium containing 1×10⁷ cells was injected subcutaneously into the left and right posterior flank regions of each mouse. The mice were housed in a pathogen-free environment and tumor growth was monitored every 3 days. Mice were killed after 21 days and the volume of each tumor was calculated according to the formula V = a×b×(a + b)/2, where “a” and “b” are respectively the length and the width of the tumor measured with a sliding caliper.

Sections (4 μm) of the xenograft tumor tissues were subjected to immunohistochemical staining as follows. Formalin-fixed and paraffin-embedded tissue sections were deparaffinized in xylene, rehydrated in a graded alcohol series, and washed with PBS. Then the sections were immersed in 10 mmol/L citrate buffer (pH 6.0) and heated in a microwave for 30 min. After cooling to room temperature, endogenous peroxidase was blocked by incubation with 3% H₂O₂ in methanol. Nonspecific binding was blocked by incubating the sections with 1% BSA in a humid chamber for 60 min. Incubation with the primary antibodies was subsequently performed overnight at 4°C using antibodies for XB130, E-cadherin, vimentin, or p-Akt. Then incubation with suitable secondary antibodies was done in PBS with 0.3% Triton X-100/5% horse serum albumin for 1 h in a humidified chamber. Detection was performed with a Dako Envision System (Dako, Glostrup, Denmark) after slides were counterstained with hematoxylin. Isotype-matched IgG (at the same dilution as the primary antibodies) was used as the negative control.

Among the 5 common human GC cell lines, we found that XB130 expression was higher in SGC7901 (a poorly-differentiated cell line) and MKN45 (a well-differentiated cell line) than in the other cell lines (Additional file 1: Figure S1A). Accordingly, we chose these two cell lines for transfection with sh-XB130. The knockdown effect of sh-XB130 was confirmed by real-time PCR (Additional file 1: Figure S1B) and Western blotting (Additional file 1: Figure S1C). Compared with Scramble shRNA-transfected cells (Scramble cells), colony formation by sh-XB130-transfected cells (sh-XB130 cells) was markedly reduced in the plate colony forming assay (Figure 1A). In addition, the number of colonies that grew in soft agar was significantly reduced by transfection of sh-XB130 (p < 0.01) (Figure 1B). When the MTT assay was used to assess cell viability over a period of 7 days, we found that viability was significantly lower in sh-XB130 cells than in Scramble cells, indicating that cell viability was suppressed by knockdown of XB130 (Figure 1C). Cell cycle analysis revealed that sh-XB130 cells were arrested in G1 phase, accompanied by a significant reduction of cells in S phase (Figure 2A). The BrdU labeling assay showed that DNA synthesis was also strongly inhibited in sh-XB130 cells (Figure 2B). These results indicate that cell proliferation was remarkably inhibited by silencing of XB130.

To assess the effect of down-regulation of XB130 on cell motility, the wound healing assay and Transwell assay were performed. After knockdown of XB130, we found that fewer cells migrated to the center of the wound in the wound healing assay (Figure 3A) or migrated into the lower chamber in the Transwell assay (p < 0.01) (Figure 3B). In addition, sh-XB130 cells were relatively smooth spheroids with few projections, while Scramble cells and Control cells developed a multipolar invasive morphology in 3D culture (Figure 3C and Additional file 2: Figure S2A). We also investigated the cell structure by staining F-actin filaments. We found that XB130 was expressed in the F-actin filaments and XB130 knockdown resulted in GC cells adopting an epithelial-like morphology (Figure 3D). These findings indicate that the motility of GC cells was suppressed along with a decrease of invasive morphologic features after down-regulation of XB130.

To determine the influence of XB130 on tumor growth in vivo, a xenograft nude mouse model was used. Colonies of sh-XB130 cells, Scamble shRNA cells, and Control SGC7901 cells were prepared. After subcutaneous injection into nude mice, all three types of cells formed tumors (Figure 4A). However, tumor growth was much slower after injection of sh-XB130 cells than after injection of Control or Scramble cells (Figure 4B). After 3 weeks, tumor volume was significantly smaller in the sh-XB130 group than in the Control and Scramble groups (p < 0.01, Figure 4B and C). These findings indicate that GC tumor growth was inhibited by downregulation of XB130.

To explore the mechanisms underlying the above-mentioned changes induced by silencing of XB130, we postulated that its downregulation might influence the expression of EMT markers and metastasis-associated proteins via the PI3K/Akt pathway. We found that knockdown of XB130 decreased the phosphorylation of Akt in xenograft GC tissues (Figure 4D) and in GC cell lines (Figure 5A). Immunofluorescence, immunohistochemistry, and Western blotting were combined to assess the expression of EMT markers. In contrast to the Scramble group, silencing of XB130 in xenograft GC tissues and cultured GC cell lines led to higher expression of the epithelial marker E-cadherin and lower expression of the mesenchymal marker vimentin (Figure 4D, Figure 5B, and Additional file 2: Figure S2B). Western blotting also showed that silencing of XB130 substantially increased the expression of epithelial markers (E-cadherin, α-catenin and β-catenin), while causing a significant decrease in the expression of mesenchymal markers (fibronectin and vimentin) and metastasis-associated proteins (MMP2, MMP9 and CD44) (Figure 5C and D).