Upregulated Vav2 in gastric cancer tissues promotes tumor invasion and metastasis

Abstract

Several studies have proved that Vav2 gene is associated with the carcinogenesis of some tumors, but the relationship between Vav2 gene and gastric cancer remains unclear. Purpose of this study is to detect the expression of Vav2 protein in gastric cancer tissues and to evaluate the clinical value of Vav2. Furthermore, both effect of Vav2 gene on invasion and metastasis of gastric cancer cells and its mechanism are investigated in vitro. Results showed that positive rate of Vav2 protein was significantly higher in gastric cancer tissues than in adjacent tissues and notably higher in metastatic lymph nodes than in gastric cancer tissues. Results of western blot were consistent with immunohistochemistry. Expression of Vav2 protein in gastric cancer tissues was related to degree of tumor differentiation, lymph node metastasis, and clinical stages. Inhibition of endogenous Vav2 in BGC823 cells led to significantly decreased cell activity, migration, and invasion ability in vitro, and expression of Rac1, MMP-2, and MMP-9 decreased, whereas expression of TIMP-1 increased. We concluded that Vav2 might promote invasion and metastasis of gastric cancer by regulating some invasion and metastasis-related genes.

Introduction

Gastric cancer, a common malignancy in East Asia, is a great health hazard associated with poor prognosis and treatment effects.^1,2 Most patients with gastric cancer suffer from poor prognosis due to the existence of metastasis, resulting from strong invasion and metastasis ability of gastric cancer cells.^3,4 Therefore, investigating genes that have regulatory functions in the process of invasion and metastasis of gastric cancer may assist to identify novel tumor markers and treatment target. Vav2, a newly discovered gene, is noticed to express abnormally in some cancers.^5–7 However, there have been hitherto few reports concerning the effect of Vav2 on metastasis of gastric cancer and the mechanism. Therefore, in this study, we investigated cancer tissues and gastric cancer cell lines, analyzed the relationship between Vav2 gene and gastric cancer invasion and metastasis, and preliminarily analyzed the molecular mechanisms. We found increased expression of Vav2 in gastric cancer, and Vav2 may promote invasion and metastasis of gastric cancer potentially by regulating invasion and metastasis-related genes, in terms of Rac1, MMP-2, MMP-9, and TIMP-1.

Materials and methods

Patients and tissue specimens

In all, 115 patients with gastric cancer in our hospital were enrolled from January 2009 to December 2010. There were 76 males and 39 females, aged 34–81 years, with a mean age of 54.617 ± 9.482 years. All patients had the primary tumor surgically removed without preoperative chemotherapy and/or radiotherapy. The specimens from patient’s primary tumors were embedded in paraffin to make 4-µm-thick serial sections for immunohistochemistry (IHC) testing. Metastatic lymph nodes in 85 cases with positive lymph node metastasis and specimens from 50 cases with normal gastric mucosa were tested by IHC. Fresh specimens of tumor tissues, metastatic lymph nodes, and adjacent normal gastric mucosa from 10 patients with advanced gastric cancer from January 2016 to May 2016 were stored at −80°C. Western blot was employed to detect expression of Vav2 protein.

Cell lines and reagents

Well-differentiated human gastric cancer cell line MKN28, moderately differentiated gastric cancer cell line SGC7901, poorly differentiated gastric cancer cell line BGC823 and AGS, and gastric epithelial cell line GES-1 were purchased from Cell Source Center of Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences. Lipofectamine™ 2000 transfection reagent was the product from Invitrogen Corporation (USA). Dulbecco’s Modified Eagle’s Medium (DMEM) and calf serum were purchased from Gibco (USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), TRIzol, fluorescence quantitative polymerase chain reaction (PCR) kit, and protein extraction kit were purchased from Sigma (USA). Gene primers and Vav2-siRNA were designed and synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. Vav2, Rac1, MMP-2, MMP-9, TIMP-1, and β-actin antibody were purchased from Santa Cruz Biotechnology (USA).

Immunohistochemical assay

After dewaxed and hydrated, paraffin-embedded specimens were treated with IHC staining. The procedure followed instructions on IHC test kit, and then, pathological section was observed by two pathological professionals blinded to this study. Five 400 magnified visual fields were selected randomly from each section, with 100 cells per visual field. The presence of yellow or brown staining in cytoplasm or the cell membrane for Vav2, Rac1, MMP-2, MMP-9, and TIMP-1 proteins was defined as positive. The positive rate was calculated with the secondary scoring method: (1)scoring based on staining intensity: colorless = 0, pale yellow = 1, brown yellow = 2, and dark brown = 3; (2) scoring according to extent of stained cells: 0% = 0, 1%–10% = 1, 11%–50% = 2, 51%–75% = 3, and 75%–100% = 4. In addition, the staining intensity score multiplied the extent of stained cells score to determine the final score. Specifically, the resulting score ≤2 was defined as negative (−) and that >2 as positive (+).

Western blotting assay

Total proteins of tissues or cells were extracted from tissues or cell samples. After protein quantification with Bradford method, 40 µg samples from each group were detected. Equal amounts of protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electotransferred onto polyvinylidene difluoride (PVDF) membrane, followed by sealing with Tris-buffered saline with Tween 20 (TBST) containing 5% skim milk powder at room temperature for 1 h. Each was incubated with diluted primary antibody at 4°C overnight. After rinsed with TBST buffer for three times, horseradish peroxidase (HRP)-labeled secondary antibodies were added for incubation at room temperature for 1 h. Chemiluminescence immunoassay (CLIA) was utilized for color development, and the bands underwent absorbance scanning. The expression of the target protein was presented as the ratio of the target protein and the internal reference protein.

Culture of gastric cell lines

Human gastric cancer cell lines MKN28, SGC7901, BGC823, and AGS and gastric epithelial cell line GES-1 were cultured in DMEM medium containing 10% fetal bovine serum. Cells at growth phase were collected for experiment.

Vav2-siRNA transfection

Based on the study by Yi et al.⁸ Vav2-siRNA sequences were designed: 5′-AAGGAGAGGTTCCTTGTTTAT-3′; non–small interfering RNA (siRNA; control siRNA) sequence: 5′-CGGCUGCAAUCGAUUGAUAGC-3′. Prior to transfection, BGC823 cells were seeded in six-well plates for 24 h with a density of 4 × 10⁵ cells/mL. The cells were rinsed with serum-free and antibiotics-free DMEM. According to the transfection reagent LipofectamineTM 2000 instructions, siRNA was transfected into gastric cell BGC823, and concentration of Vav2-siRNA was kept as 40 µmol/L, and then, results were detected 24 h after transfection.

MTT assay

BGC823 cells were seeded in 96-well plates at the density of 5 × 10⁴ cells/mL. Cells at a confluency of 70%–80% were transfected with Vav2-siRNA or control siRNA for 20 h. Six replicate wells were added in each group. Subsequently, 20 µL (5 mg mL) of MTT was added for incubation for 4 h. The medium was then discarded. A volume of 150 µL dimethyl sulfoxide (DMSO) was added to each well and shaken at room temperature for 15 min, and microplate reader was used to measure absorbance value (A value) at a wavelength of 490 nm. The experiment was conducted three times independently.

Cell scratch assay

BGC823 cells were digested to derive single-cell suspension, and those at the concentration of 1 × 10⁶ cells/mL were seeded into six-well plates. Cells at a confluency of 60%–70% were transfected with Vav2-siRNA or control siRNA. While cells grew to 100% confluency, the medium was discarded and cells were rinsed with phosphate-buffered saline (PBS). A sterile pipette tip was used to make a central linear wound at the bottom of six-well plates, and scraped cells were washed with PBS. The speed of wound healing was then observed under microscope. The experiment was repeated for three times.

Transwell assay

Transwell chamber was coated with 100 µL of Matrigel for ultraviolet radiation. The digested BGC823 cells were seeded in six-well plates at a density of 1 × 10⁶ cells/mL. Cells at a confluency of 60%–70% were transfected with Vav2-siRNA or control siRNA for 20 h, respectively. Subsequently, 200 µL cells from each group were seeded in the upper chamber and DMEM was added to the lower chamber. After 24 h, Matrigel glue and excess BGC823 cells in the upper chamber were wiped with cotton swabs. Methanol was used to fix the membranes for 10 min. The cells penetrating to the lower membrane were counted after crystal violet staining. Each experiment was performed three times.

Fluorescence quantitative PCR

One-step method was used to extract total RNA of BGC823 cells. Integrity, purity, and content of RNA were identified. An amount of 2 µg of total RNA was reverse transcribed to synthesize complementary DNA (cDNA). PCR was performed to detect relative expression levels of messenger RNA (mRNA) in Vav2, Rac1, MMP-2, MMP-9, and TIMP-1. 2^−ΔΔCt was used for calculation. Primer sequences were as follows—Vav2: 5′-TCAAAGAAGCACTGGAAGCC-3′, 5′-CGAAAAAACTGGAAGCCCTG-3′; Rac1: 5′-CCCTATCCTATCCGCAAACA-3′, 5′-CGCACCTCAGGATACCACTT-3′; MMP-2: 5′-CAGGAGGAGAAGGCTGTGTT-3′, 5′-AGGGTGCTGGCTGAGTAGAT-3′; MMP-9: 5′-CCTTCTACGGCCACTACTGT-3′, 5′-TCCACCTGGTTCAACTCACT-3′; TIMP-1: 5′-ACTTCCACAGGTCCCACAAC-3′, 5′-GCATTCCTCACAGCCAACAG-3′; and glyceraldehyde 3-phosphate dehydrogenase (GAPDH): 5′-GACCCCTTCATTGACCTCAAC-3′, 5′-CGCTCCTGGAAGATGGTGAT-3′.

Statistical analysis

SPSS19.0 software was used for statistical analysis. Measurement data were expressed as mean ± standard deviation ( $\overline{X}$ ± s). The between-group differences were analyzed with the t-test and analysis of variance (ANOVA). Count data were expressed as rate or percentage and examined with the chi-square (χ²) test. A p value <0.05 indicated a significant difference.

Results

Expression of Vav2 protein in gastric cancer tissues, metastatic lymph node tissues, and normal gastric mucosa (IHC)

IHC showed that positive rate of Vav2 protein in metastatic lymph node was 87.059% (74/85), followed by 69.565% (80/115) in gastric cancer tissues, and the lowest rate of 18.000% (9/50) in normal gastric mucosa. The positive rate in the three groups was significantly different (χ² = 67.959, p < 0.001; Figure 1(a)). Results of western blot were in accordance with IHC. Figure 1(b).

Figure 1.

Vav2 expression in gastric cancer tissues, metastatic lymph node tissues, and normal gastric mucosa. (a) Clinical samples of tissues embedded in paraffin were applied to detect Vav2 protein with immunohistochemistry (IHC 400×), and staining location was in cytoplasm or the cell membrane. (b and c) Expressions of Vav2 protein in fresh tissues of gastric cancer tissues, metastatic lymph node tissues, and normal gastric mucosa were tested with western blotting, and protein expression level was also shown (*p < 0.05 versus normal gastric mucosa; ^#p < 0.05 versus gastric cancer tissues). Highest protein Vav2 level was found in metastatic lymph node tissues.

[Figure omitted. See PDF]

The relationship between Vav2 expression in gastric cancer tissues and clinicopathological features (IHC)

Results revealed that Vav2 protein was related to differentiation, lymphatic metastasis, and tumor–node–metastasis (TNM) stages (p < 0.05). Higher positive rate of Vav2 protein was detected in poorly differentiated tissues, metastatic lymph nodes, and advanced TNM stages (all p < 0.05). Vav2 protein was not significantly associated with other pathological parameters (p > 0.05; Table 1).

Table 1.

Relationship between Vav2 protein and clinicopathological parameters in gastric cancer patients (n = 115).

Clinicopathological parameters	Positive (80)	Negative (35)	χ2	p
Gender
Male (76)	49	27	2.744	0.098
Female (39)	31	8
Age (years)
≥60 (32)	24	8	0.619	0.432
<60 (83)	56	27
Tumor differentiation
Highly/moderately (81)	51	30	5.641	0.018
Poorly/undifferentiated (34)	29	5
Depth of invasion
T1/T2 (35)	26	9	0.530	0.467
T3/T4 (80)	54	26
Lymphatic metastasis
Positive (82)	62	20	4.931	0.026
Negative (33)	18	15
Nerve/vessel invaded
Invaded (57)	42	15	0.906	0.341
Not invaded (58)	38	20
TNM stages
I–II (38)	21	17	5.483	0.019
III–IV (77)	59	18

TNM: tumor–node–metastasis.

Expression of Rac1, MMP-2, MMP-9, and TIMP-1 proteins in gastric cancer tissues and normal gastric mucosa and their relationship with Vav2 protein (IHC)

Results of IHC showed that positive rate of Rac1 protein was 56.522% (65/115), which was higher than that in normal gastric mucosa (28.000%, 14/50; χ² = 11.360, p < 0.001). Also, significant difference between gastric cancer tissues and normal gastric mucosa was verified about MMP-2 and MMP-9 proteins (p < 0.01), whereas no significant difference was found between gastric cancer tissues and normal gastric mucosa about TIMP-1 protein (p > 0.05; Table 2, Figure 2). Result of Spearman showed that expression of Vav2 was positively related to Rac1, MMP-2, and MMP-9 (r = 0.5022, p < 0.001; r = 0.4136, p < 0.001; r = 0.2005, p = 0.0317), and negative correlation was detected between expression of Vav2 and TIMP-1 (r = −0.3206, p < 0.001).

Table 2.

Expression of Rac1, MMP-2, MMP-9, and TIMP-1 proteins in gastric cancer and normal gastric mucosa (n = 115).

	Rac1		MMP-2		MMP-9		TIMP-1
	Negative	Positive	Negative	Positive	Negative	Positive	Negative	Positive
Gastric cancer	50	65	31	84	37	78	73	42
Normal gastric mucosa	36	14	29	21	31	19	36	14
χ2	11.360		14.513		12.796		1.129
p	<0.001		<0.001		<0.001		0.288

Figure 2.

Rac1, MMP-2, MMP-9, and TIMP-1 expression in gastric cancer tissues and normal gastric mucosa (IHC 400×). Clinical paraffin samples of tissues were applied to detect Rac1, MMP-2, MMP-9, and TIMP-1 proteins with IHC, and staining location was in cytoplasm or cell membrane. (a and b) Expressions of Rac1 protein in gastric cancer tissues and normal gastric mucosa. (c and d) Expressions of MMP-2 protein in gastric cancer tissues and normal gastric mucosa. (e and f) Expressions of MMP-9 protein in gastric cancer tissues and normal gastric mucosa. (g and h) Expressions of TIMP-1 protein in gastric cancer tissues and normal gastric mucosa.

[Figure omitted. See PDF]

The expression of Vav2 protein in gastric cell lines (western blot)

Result of western blot demonstrated that Vav2 protein expression was significantly higher in three gastric cancer cell lines than in gastric epithelial cell line GES-1 (p < 0.01), with the strongest expression in BGC823 (p < 0.01). Therefore, BGC823 cells were selected in the subsequent experiment (Figure 3).

Figure 3.

Vav2 expression in gastric cell lines. (a) Gastric cell lines MKN28, SGC7901, BGC823, AGS, and GES-1 were subjected to western blot assay to determine the protein levels of Vav2. (b) Values were shown as mean ± SD (n = 4 in each group; *p < 0.05 versus GES-1 group; ^#p < 0.05 versus MKN28, SGC7901, and AGS group). Highest protein Vav2 level was found in BGC823 cells.

[Figure omitted. See PDF]

The effect of Vav2-siRNA transfection on expression of Vav2 in BGC823 cells

Western blot revealed that 48 h after 40 µmol/L Vav2-siRNA was transfected with BGC823 cells, Vav2 protein in cells was significantly lower compared with the control group (transfected with non-siRNA) and the blank group (only transfected with LipofectamineTM 2000; p < 0.01; Figure 4).

Figure 4.

Effect of Vav2-siRNA for Vav2 regulation in BGC823 cells. BGC823 cells were transfected with Vav2-siRNA (40 µmol/L), non-siRNA (control group), or Lipofectamine™ 2000 (blank group), alternatively. (a) After 48 h, the expressions of Vav2 were identified by western blot. (b) The expression levels of Vav2-siRNA (40 µmol/L), non-siRNA (control group), and Lipofectamine™ 2000 (blank group; *p < 0.05 vs control group).

[Figure omitted. See PDF]

The effect of Vav2-siRNA transfection on BGC823 cell activity

Cell activity was significantly lower in Vav2-siRNA group than in the control group and the blank group (p < 0.01; Figure 5) 48 h after 40 µmol/L Vav2-siRNA was transfected with BGC823 cells.

Figure 5.

Effects of Vav2-siRNA on the activity of BGC823 cells (MTT assay). Cells transfected with Vav2-siRNA, non-siRNA, and Lipofectamine™ 2000 were subjected to MTT assay to show activity of BGC823 cells after treated with Vav2-siRNA. The results showed that activity of BGC823 cells in Vav2-siRNA group was lower than in control group in a time-dependent manner (*p < 0.05 vs control group).

[Figure omitted. See PDF]

The effect of Vav2-siRNA transfection on migration and invasion abilities of BGC823 cells

After Vav2-siRNA was transfected with BGC823 cells, scratch test and Transwell assay found that migration and invasion abilities of BGC823 cells transfected with Vav2-siRNA were significantly lower, as compared with the control group and the blank group (p < 0.05), and no significant difference was found between the negative control group and the blank control group (p > 0.05; Figure 6).

Figure 6.

Effects of Vav2-siRNA on the migration and invasion of gastric cancer cell line BGC823 (scratch test and Transwell assay). BGC823 cells transfected with Vav2-siRNA, non-siRNA, and Lipofectamine™ 2000 were also tested by (a) scratch test and (b) transwell assay (*p < 0.05 vs control group).

[Figure omitted. See PDF]

The effect of Vav2-siRNA transfection on expression of Rac1, MMP-2, MMP-9, and TIMP-1 in BGC823 cells

mRNA and protein expression of Rac1, MMP-2 and MMP-9 were downregulated 48 h after BGC823 cell transfected with 40 µmol/L Vav2-siRNA, whereas those in TIMP-1 was upregulated (p < 0.01). Gene expression showed no significant difference in the control group and the blank group (p > 0.05; Figure 7).

Figure 7.

Effects of inhibiting endogenous Vav2 on expression of Rac1, MMP-2, MMP-9, and TIMP-1 in BGC823 cells. Cells transfected with Vav2-siRNA, non-siRNA, and Lipofectamine™ 2000 were then subjected to (a) QPCR and (b) western-blot assay to detect the mRNA or protein expression levels (*p < 0.05 vs control group).

[Figure omitted. See PDF]

Discussion

Patients with gastric cancer have poor prognosis, and 5-year survival rate of patients with advanced gastric cancer is less than 40%.⁹ Despite great advances in diagnosis and treatment,^10–12 the long-term therapeutic effect of gastric cancer is far from satisfactory. The main cause of poor prognosis is strong invasion and migration ability of gastric cancer cells,^13–16 which contributes to metastasis at early-stage gastric cancer; additionally, further changes will occur in tumor cells’ metastasis process,^17,18 resulting in treatment failure, thereby causing gastric cancer recurrence and further metastasis. Therefore, it may be of great significance to determine the genes that play an important role in the invasion and metastasis of gastric cancer. Vav2 is a recently discovered gene closely associated with tumor. Studies have found that Vav2 gene is associated with breast cancer, liver cancer, lung cancer, and T lymphocyte leukemia.^19–22 According to this study, IHC and western blot confirmed that expression of Vav2 protein was enhanced in the order of normal gastric mucosa, gastric cancer tissue, and metastatic lymph nodes, suggesting Vav2 might be associated with occurrences and progression of gastric cancer. Our study verified that Vav2 protein expression in gastric cancer was related to degree of tumor differentiation, lymph node metastasis, and TNM stages. Vav2 expression was stronger in gastric cancer tissues with poor differentiation, lymph node metastasis, and advanced TNM stages. The results revealed that Vav2 protein might be involved in gastric cancer progression and metastasis. The study also found that Vav2 protein expression was higher in metastatic lymph node tissues than in the primary tumor, indicating increased Vav2 protein expression promoted the metastasis of gastric cancer in the process of metastasis. These results indicate that Vav2 expression in gastric cancer tissues has an important clinical significance, and Vav2 possibly serves as a novel tumor marker for comprehensive diagnosis and target for treatment to gastric cancer.

To understand the role Vav2 gene plays in invasion and metastasis of gastric cancer cells, in vitro experiments were carried out in our study. Based on western blot screening, we observed the strongest expression of Vav2 in BGC823, which was derived from the poorly differentiated adenocarcinoma and was utilized in our study in vitro to investigate the function of Vav2 gene. It was found that inhibition of endogenous gene Vav2 in BGC823 cells contributed to decreased activity of tumor cells and notably reduced invasion and migration abilities. This indicated that Vav2 gene promoted invasion and migration of gastric cancer cells; therefore, inhibition of Vav2 gene expression led to inhibited tumor progression. Studies have revealed that Vav2/Rac1 pathway can promote the progression of tumors.^23,24 This study detected the expression of Rac1 after inhibition of Vav2. It was demonstrated that Rac1 expression was decreased due to inhibited Vav2, indicating Vav2/Rac1 pathway promoted the invasion and metastasis of gastric cancer, which was consistent with findings in other studies.^25,26

For preliminary understanding of mechanism of Vav2 gene’s involvement in the gastric cancer invasion and migration, this study examined expression changes in tumor invasion and migration-related genes before and after inhibition of Vav2 gene, such as matrix metalloproteinase-2 (MMP-2), MMP-9, and tissue inhibitor of metalloproteinase-1 (TIMP-1). MMPs-TIMPs, as an important gene family, affect tumor invasion and metastasis, including some vital members, such as MMP-2, MMP-9, and TIMP-1. MMP-2 and MMP-9 play a significant role in promoting the invasion and migration of gastric cancer cells, whereas TIMP-1 inhibits this process by regulating MMP-9.^27–32 This study showed that inhibition of Vav2 in BGC823 cells leads to markedly reduced expression of MMP-2 and MMP-9 and significantly increased expression of TIMP-1, suggesting that Vav2 gene in gastric cancer cells may be involved in invasion and metastasis of gastric cancer cells by regulating MMP-2, MMP-9, and TIMP-1. However, the molecular mechanism needs further study.

This study revealed that increased expression of Vav2 was associated with gastric cancer. In vitro studies have shown that Vav2/Rac1 pathway may accelerate the invasion and metastasis of gastric cancer, and its mechanisms are associated with regulation of MMPs-TIMPs family members. But clinical research in our study was a single-center study, and multi-center study is needed to obtain precise results. Moreover, we only conducted experiments in vitro. Therefore, experiments in vivo should be conducted in the future to determine the role that Vav2 has in gastric cancer invasion and metastasis.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The author(s) received no financial support for the research, authorship, and/or publication of this article.