| BACKGROUNDGastric cancer is the most common malignant tumors of the digestive system, the morbidity and mortality are very high. Invasion, metastasis and recurrence is the leading cause of death of most patients with gastric cancer. Epithelial-mesenchymal transition (epithelial mesenchymal transition, EMT) provides the basis for the invasion and metastasis of tumor cells of epithelial origin. EMT is a key early events induced tumor invasion and metastasis. The role of EMT in cancer invasion and metastasis become a research focus. Study of gastric cancer invasion and EMT mechanism can help to find therapeutic targets. A variety of signal transduction pathways involved in the EMT of tumor cells, such as transforming growth factor-β (TGF-β), MAPK, Notch and Wnt pathway. When the Wnt/β-catenin pathway is abnormally activated, the adhesion between the tumor cells are weakened, inducing the EMT process of tumor cells, enhancing the ability of tumor cell invasion and metastasis. Eph receptors have been recognized as the largest family of RTKs, which plays a key role in the regulation of tumor cell growth, proliferation,metastasis-related signal pathway. EphA2is one of the14Eph receptors, which is high expressed in many tumors. Recent studies have found that EphA2is related to EMT, but the mechanism is unclear. Further investigations to the mechanism for EphA2regulation of EMT can help to guide the postoperative treatment of gastric cancer.OBJECTIVETo investigate the effect and mechanism of EphA2in gastric cancer EMT. METHODS1. The expression of EphA2and EMT related proteins E-cadherin, β-catenin and vimentin in158specimens of gastric cancer tissues and specimens of paraneoplastic non-cancerous gastric mucosa tissues was detected by immunohistochemistry (IHC). The relationship between their expression and clinicopathological features, prognosis was statistically analyzed. The expression of EphA2mRNA of gastric cancer cells was determined by real-time reverse transcription-poly-merase chain reaction (RT-PCR).2. Gasrtic cancer AGS cells were treated with Lithium chloride (LiCl), cell proliferation was assessed using MTT assay. The expression of GSK-3β and p-GSK-3βSer9was measured by Western blot. The expression of β-catenin was detected by immunofluorescence(IF). The expression of c-myc mRNA and protein was detected by real time RT-PCR and Western blot.3. The specific EphA2small interference RNA (siRNA) was transfected into gastric cancer AGS cells using lipofectamine2000. The expression of EphA2mRNA and protein was detected by real time RT-PCR and Western blot. Gasrtic cancer AGS cells were treated with both LiCl and EphA2siRNA, the expression of GSK-3β, p-GSK-3βSer9, β-catenin and c-myc were determined.4. LiCl was used to stimulate EMT in gastric cancer AGS cells, the expression of EMT related markers E-cadherin, β-catenin and vimentin mRNA and protein was detected by real time RT-PCR and Western blot, and the cell cycle phase distribution was analyzed by flow cytometry, the invasive ability was determined by Transwell invasion assay. siRNA was used to silence the EphA2expression, the expression of E-cadherin, β-catenin and vimentin mRNA and protein was detected by real time RT-PCR and Western blot, the cell cycle phase distribution was analyzed by flow cytometry, the invasive ability was determined by Transwell invasion assay. Gasrtic cancer AGS cells were treated with both LiCl and EphA2siRNA, the expression of E-cadherin, β-catenin and vimentin mRNA and protein was detected by real time RT-PCR and Western blot, the cell cycle phase distribution was analyzed by flow cytometry, the invasive ability was determined by Transwell invasion assay.RESULTS1. The expression of EphA2and vimentin in gastric cancer tissues was significantly higher than that in paraneoplastic non-cancerous gastric mucosa tissues (P<0.01). The expression of E-cadherin in gastric cancer tissues was significantly lower than that in paraneoplastic non-cancerous gastric mucosa tissues (P<0.01). The nuclear expression of β-catenin in gastric cancer tissues was significantly higher than that in paraneoplastic non-cancerous gastric mucosa tissues (P<0.01). Overexpression of EphA2was statistically significantly associated with depth of invasion, TNM stage and lymph node metastasis (P<0.05). Down-regulated expression of the epithelial protein E-cadherin, overexpression of the mesenchymal protein vimentin and nuclear expression of P-catenin were associated with the depth of tumor invasion, tumor differentiation, TNM stages and lymph nodemetastasis (P<0.05). The Spearman rank test indicated that the positive expression of EphA2was negatively associated with E-cadherin expression(r=-0.625, P<0.01) and was positively correlated with β-catenin nuclear expression(r=0.375, P<0.01) and vimentin expression(r=0.330, P<0.01). Univariate analysis showed that the overexpression of EphA2and vimentin, nuclear expression of β-catenin and down-regulation of E-cadherin indicate a poor outcome (P<0.05). Moreover, multivariate Cox analysis showed that EphA2expression, E-cadherin expression and β-catenin nuclear expression were independent prognostic factors for postoperative gastric cancer(P<0.05).2. The cell proliferation was significantly different in gastric cancer AGS cells treated with different concentrations of LiCl at different times(.P <0.05), and the best concentration is20mmol/L, the optimal time is24hours. Compared to control group, the expression of p-GSK3-βSer9protein level in group added with LiCl was increased significantly (P<0.05). The nuclear expression of (3-catenin in experimental group was significantly higher than control group(P>0.05). The expression of c-myc mRNA and protein level in experimental group were increased significantly (P<0.05).3. Compared to blank and negative control group, the mRNA and protein level of EphA2in EphA2siRNA group were decreased significantly(P<0.05). Compared to the LiC1group, EphA2siRNA could significantly decrease the expression of p-GSK3-βSer9, the nuclear expression of (3-catenin and the expression of c-myc(P<0.05).4. Added LiC1to gastric cancer AGS cells induced the decrease of the E-cadherin mRNA and protein expression(P<0.05), the increase of the β-catenin mRNA and protein expressio(P<0.05), and the increase of the vimentin mRNA and protein expression(P<0.05), the number of cells in the G0/G1phase were significantly decreased(P<0.05), the number of cells of in the S phase and G2/M phase were significantly increased (P<0.05), the invasion of gastric cancer AGS cells were significantly increased(P<0.05). In EphA2siRNA group, the mRNA and protein level of E-cadherin were significantly increased (P<0.05), the mRNA and protein level of β-catenin,vimentin and c-myc were significantly decreased(P<0.05), the number of cells in the G0/G1 phase were significantly increased(P<0.05), the number of cells of in the S phase and G2/M phase were significantly decreased (P<0.05), the invasion of gastric cancer AGS cells were significantly decreased(P<0.05). Gasrtic cancer AGS cells were treated with both LiCl and EphA2siRNA, compared to the LiCl group, the mRNA and protein level of E-cadherin were significantly increased (P<0.05), the mRNA and protein level of β-catenin vimentin and c-myc were significantly decreased(P<0.05), the number of cells in the G0/G1phase were significantly increased(P<0.05), the number of cells of in the S phase and G2/M phase were significantly decreased (P<0.05), the invasion of gastric cancer AGS cells were significantly decreased(P<0.05).CONCLUSION1. The expression of EphA2and EMT related proteins are closely related with progression and metastasis of gastric cancer, and might be regarded as factors of poor prognosis in gastric cancer. EphA2is higher expressed in gastric cancer AGS cells.2. EphA2siRNA led to the inhibition of EphA2expression, and inverse the activation of Wnt/(3-catenin signal pathway induced by LiCl.3. Silencing EphA2expression could inverse EMT and cell invasion induced by LiC1in gastric cancer AGS cells.
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