| BACKGROUND:Gastric cancer remains one of the most common malignancies. The current best approach for treating gastric cancer is the complete surgical removal of the tumor with the adjacent lymph nodes. However, the efficacy of this therapeutic approach as well as chemotherapy, hormone and radiotherapy is very limited. The prognosis of gastric cancer is poor with an estimated 5-years survival rate of less than 25% worldwide. Invasion, metastasis and recurrence are the major causes of gastric cancer patients'death and the key factors affecting clinical treatment and prognosis. However, the mechanisms of gastric cancer development, invasion and metastasis remain unclear and therefore further investigation is of consequence. Receptor tyrosine kinases (RTKs), the common products of transforming oncogenes, have been widely used as indicators in the genesis and progression of human tumors. Until now, the erythropoietin-producing human hepatocellular (Eph) receptors have been recognized as the largest family of RTKs, which contains 14 distinct receptors including eight identified ligands. In Eph family, EphA2 that locates on human chromosome 1p36.1 is expressed at a lowest level in epithelial cells. However, such expression increased in carcinoma cells, and the functional alterations of Eph receptor kinases are prevalent in many tumors. It's reported that high EphA2 expression level was correlated with the tumor metastasis and poor prognosis. Increased expression of EphA2 can promoted tumor growth and enhanced the metastatic potential. However, the underlying mechanisms for EphA2 promote tumor invasion are still unclear. Matrix metalloproteinase-9 (MMP-9), one member of matrix metalloproteinases, has been paid much attention because it induces cancer metastasis by degrading type IV collagen, a major constituent of vascular basement membrane. Now, MMP-9 is considered to be important in facilitating tumor invasion and spread. It is reported that there was a positive correlation between EphA2 and MMP-9 expressions in hepatocellular carcinoma and ovarian cancer tissues. However, whether EphA2 promote tumor invasion through regulation of MMP-9 is still unclear and need further investigation.OBJECTIVE:To investigate the role of EphA2 in gastric cancer invasion, and the possible mechanisms for EphA2 promote gastric cancer invasion.METHODS:1. The expression of EphA2 and MMP-9 in 97 specimens of gastric cancer tissues and 24 specimens of normal gastric mucosa tissues was detected by immunohistochemistry (IHC). The relationship between their expression and clinicopathological features, prognosis was statistically analyzed.2. The specific EphA2 small interference RNA (siRNA) plasmid was constructed and transfected into gastric cancer SGC-7901 cells using lipofectamine 2000. Three groups of cells were used in the in vitro studies, including black control group (untransfected cells), negative control group (cells transfected with the non-silencing siRNA) and EphA2 siRNA group (cells transfected with the EphA2 siRNA). The expression of EphA2 mRNA was detected by real-time reverse transcription-polymerase chain reaction (RT-PCR). The expression of EphA2 protein was measured by western blot. Cell proliferation was assessed using methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. The cell cycle phase distribution was determined by flow cytometry. Transwell analysis was done to measure the invasion of SGC-7901 cells. The expression of MMP-9 was also detected by real-time RT-PCR and western blot.3. Suspensions of SGC-7901 tumor cells were implanted in the subcutaneous of nude mice. The animals were randomly divided into three groups (n=6) that received the following treatment:black control group (0.9% sodium chloride intratumorally), negative control group (non-silencing siRNA plasmid+liposome intratumorally) and the EphA2 siRNA group (EphA2 siRNA plasmid+liposome intratumorally). The volume of each tumor was measured once a week. Three weeks after the treatment of therapy, the mice were sacrificed and the weight of the tumor was recorded. The expression of EphA2 and MMP-9 protein in tumor xenograft was detected by IHC.RESULTS:1. The expression of EphA2 and MMP-9 in gastric cancer tissues was significantly higher than that in normal gastric mucosa tissues (P<0.01). Overexpression of EphA2 and MMP-9 was statistically significantly associated with the size of tumor, depth of invasion, TNM stage and numbers of lymph node metastasis (P＜0.05), moreover a significant correlation was observed between MMP-9 expression and histologic type (P=0.029). Next, the expression of EphA2 and MMP-9 was significantly positive correlation in gastric cancer tissues (r=0.732, P<0.01). In univariate analysis, elevated MMP-9 expression was associated with 5-year survival rate (P=0.037). Moreover, the impact of MMP-9 expression on 5-year survival rate was statistically significant (P=0.019). In Cox multivariate analysis, expression of EphA2 and MMP-9 immunostaining was all found to be independent prognostic factors (P＜0.05).2. EphA2 siRNA plasmid was constructed successfully and transfected into the SGC-7901 cells efficiently. Compared to negative control group, the mRNA and protein level of EphA2 in EphA2 siRNA group were reduced by 73.4% and 65.1%(P<0.01), respectively. The growth of cells was significantly decreased in EphA2 siRNA group compare to black control group or negative control group (P<0.01). Compared to black control group or negative control group, specific EphA2 siRNA significantly increased the number of cells in the G0/G1 phase (P<0.01), significantly decreased the number of cells of in the S phase (P＜0.05) and G2/M phase (P<0.05), and significantly decreased the proliferation index (P<0.01). The migration and invasion of SGC-7901 cells were significantly decreased in the cells transfected with the EphA2 siRNA compare to the cells transfected with the non-silencing siRNA or untransfected cells (P<0.01). In the meantime, the expression of MMP-9 mRNA and protein level in EphA2 siRNA group were decreased significantly compared to black control group or negative control group (P＜0.01). 3. The model of gastric cancer SGC-7901 cell in subcutaneous of nude mice was established. Both the volume and the weight of the tumors in EphA2 siRNA group were significantly decreased compared to black control group (P<0.01) or negative control group (P＜0.01). The rate of tumor inhibition in the EphA2 siRNA treated xenograft tumors volume was approximately 47.5%. The expression of EphA2 and MMP-9 protein was higher in the black control group or negative control group compared to the EphA2 siRNA group.CONCLUSION:1. Overexpression of EphA2 and MMP-9 is closely related with progression and metastasis of gastric cancer, and might be regarded as factors of poor prognosis in gastric cancer.2. EphA2 siRNA led to the efficient and specific inhibition of EphA2 expression in gastric cancer SGC-7901 cells. Silencing of EphA2 expression inhibits the proliferation, migration, invasion and expression of MMP-9 in SGC-7901 cells.3. Intratumoral injection with EphA2 siRNA plasmid inhibits the growth, invasion of SGC-7901 tumor xenografts and suppresses the expression of EphA2 and MMP-9 protein. |
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