Biological Significance Of EphA2 Expression In Gastric Carcinoma And The Researching In Vitro

Objectives: Gastric carcinoma, a leader compared with other malignant tumor, is one of the most common malignant tumors. Recently years research shows that the genesis and development of gastric carcinoma is a process involving multistep and multi-gene. Therefore, it is extremely important to investigate pathogenesis and search new target of prevention and therapy and effective drug in gastric carcinoma for decreased morbidity and mortality.Receptor tyrosine kinase (RTK) transmits extracellular signals to nuclear, and then initiates a cascade of signaling events that modulate cellular responses.The EPH (erythropoietin-producing hepatocellular) gene family represents the largest known family of receptor tyrosine kinases. EphA2 was the 3rd found and cloned out the whole long of cDNA and was the first gene with tyrosine kinases activity in EPH family. In normal cells, EphA2 appears to be restricted to intercellular junctions between epithelial cells, where it binds ligands, anchored to the membrane of adjacent cells, to be a complex, which then initiates tyrosine kinases in cytoplasm and participates in embryonic development, cell migration and vasiformation and so on. Normal EphA2 expression signal weaken Ras/MAPK cascade reaction, which activated by PDEF, EGF, VEGF, and restrain cell growth, then negatively regulate cellular adherin through breaking spot-adherin. EphA2 was paid more and more attention in carcinogenesis and development in recent years. It's found that EphA2 is overexpressed in many human cancers such as breast, colon, prostate and nonsmall-cell lung cancer (NSCL), whose growth, transformation and metastasis correlate with EphA2 mutation, over expression, abnormal location and (or) abnormal inactivation. Above all, EphA2 is a new target for clinical diagnosis and therapy, while there is not exactly experimental basement for EphA2 abormal expression in gastronoma.Geldanamycin (GA), benzoquinoid ansamycin antibiotic, targets heat shock protein 90 (Hsp90), is an inhibitor for Hsp90. Hsp90 is a group of highly conservative molecular chaperones, which is widely found in organism. EphA2, abnormally highly overexpresses in tumor, whose downstream proteins take part in key point of tumor progression. There are a lot of Hsp90 downstream proteins including steroid receptors, therosine kinase receptors and ammonia kinase receptors etc. EphA2, as a member of RTK family, is probably a downstream protein of Hsp90. In recent years'research, GA has become a novel target for anticancer drug development because it is a potent inhibitor of many kinds of tumor and it can induce apoptosis. However, tumor-suppress effect of GA on gastric carcinoma has never been documented, and it is unknown that if GA achieves its way of cellular cycle-modulation and apoptosis-induction through the expression of EphA2 protein.RNA interference (RNAi) is a sequence-specific and post-transcriptional gene silencing process mediated by double-stranded RNAs (dsRNA) through direct mRNA degradation. Currently, RNAi technology is not only a powerful tool to exploit function of human genome and identify target of drug, but also holds great promise in the field of clinical applications.Therefore, our investigation was following: significance of EphA2 and its ligand EphrinA1 in carcinogenesis and progression of gastric carcinoma, and the relationship between these proteins expression in gastric carcinoma; relationship between EphA2 expression and adherin, proliferation, apoptosis and angiogenesis in gastric carcinoma; effect of GA, inhibitor of Hsp90, on proliferation, apoptosis of human gastric carcinoma cells-MGC803; effect of GA on expression of EphA2, Survivin and Caspase-3 protein, further investigation for whether EphA2 was a target of GA on human gastric carcinoma therapy; silencing EphA2 expression by RNAi, and then testing proliferation on human gastric carcinoma cell line MGC-803 contributed to explaining the relation between EphA2 expression and carcinogenesis in gastric carcinoma. It may provide theoretical evidence for whether EphA2 is a new target for preventing and treating gastric carcinoma. It may establish fundament for highlighting mechanism of carcinogenesis and progression of gastric carcinoma as well.Methods:1 Studies on EphA2 and its ligand EphrinA1 expression in gastric carcinoma, paired adjacent mucosa to gastric carcinoma and normal gastric mucosa at surgical margin of gastric carcinomaEphA2, EphrinA and E-cadherin proteins expression were detected by means of immunohisochemistory (IHC) in 82 cases surgical resected primary gastric carcinoma tissues, paired adjacent mucosa (2~5 cm from margin of gastric carcinoma ) and normal mucosa at surgical margin (at least 5cm from margin of gastric carcinoma and histologically proven) tissues, and then analyzed the relation between their expression and clinic charactors; EphA2 mRNA and protein expression were detected in 30 cases drawn from above 82 cases randomly by RT-PCR and Western Blot.Neovasculature was detected using immunohistochemistry labeled with CD34 antibody and indicated by microvessel density (MVD) in 82 tissues of gastric carcinoma, analyzing the correlation between EphA2 and MVD, between these proteins and clinic characters of gastric carcinoma. Apoptosis rate and proliferative index (PI) were detected in 82 cases by FCM, and then the relation between EphA2 expression and apoptosis, proliferation was analyzed.2 Effect of GA on proliferation and apoptosis of human gastric carcinoma cells-MGC803 and its regulation mechanism to EphA2Human gastric carcinoma cell line MGC-803 was conventional cultured. Effect of GA (20, 40, 200, 400, 2000 nmol/L) for 12h, 24h, 48h and 72h on proliferation suppressions of MGC-803 cells were detected by MTT assay; cell-cycle change and apoptosis rate were detected by FCM; cell morphology change was observed by Giemsa staining; expression of EphA2, Survivin and Caspase-3 protein detected by IHC and FCM.3 Effect of RNAi silencing EphA2 on proliferation and apoptosis of human gastric carcinoma cell line MGC-803Three locations targeted were designed and three plasmid vectors (S1, S2 and S3) expressed shRNAs for silencing EphA2 were constructed according to transcription RNA location of human of EphA2 gene sequence and designing principle of small interference RNA (siRNA) by Wuhan JingSai Company. Also a negative control plasmid vector HK was constructed. These plasmid vectors can express green fluorescence protein (GFP) and resist neomycin (neo). They were transfected into human gastric carcinoma MGC-803 cells by cations liposome vector. The transfection efficiency of plasmid vector was evaluated by calculating the ratio of fluorescent cells to total cells tested by FCM and Laser confocol scan microscope to select the most suitable ratio of cations liposome vector to plasmid vector; the best transfection time was selected by MTT and used in later experiment; EphA2 mRNA and protein of MGC-803 cells were detected by RT-PCR and Western blot 48h after transfection, respectively, and the best plasmid vector silencing EphA2 of MGC-803 cells was selected and used in later experiment; Changes of EphA2 protein, proliferative activity, cell cycle and apoptosis were detected in MGC-803 cells transfected with the best plasmid vector silencing EphA2.Results:1 EphA2 and its ligand EphrinA1 protein expression in various lesions of gastric tissues and relationship between clinic charactersEphA2 and EphrinA1 protein expression rates shown by IHC: higher in carcinoma than in adjacent group and normal group (P<0.01); higher in medium and low differentiation than in well differentiation (P<0.05); higher in deeper infiltration than in superficial layer (P<0.05); higher in lymph node metastasis than in non-lymph node metastasis (P<0.05). E-cadherin expression rates: lower in carcinoma than in adjacent group and normal group (P<0.01); lower in medium and low differentiation than in well differentiation (P<0.05); lower in deeper infiltration than in superficial layer (P<0.05); lower in lymph node metastasis than in non-lymph node metastasis (P<0.05). EphA2 expression was positively correlated with EphrinA1 significantly (r=0.707, P<0.01); above two protein were negatively correlated with E-cadherin significantly (r=-0.231, r=-0.403, P<0.01). There is no difference of EphA2 mRNA among every group shown by RT-PCR (P>0.05), while Western Blot results were identical with IHC. Apoptosis rate was higher in EphA2 high-expression group than in low-expression (P=0.018) and PI was contrary (P=0.002) shown by FCM.MVD was significantly higher in gastric carcinoma than in adjacent and normal tissues (P<0.01); MVD was correlation with differentiation, infiltration depth, lymph node metastasis and diameter of tumor (P<0.05); EphA2 protein expression was positively related with MVD significantly (r= 0.485, P<0.01).2 Proliferative and apoptotic effect of GA on human gastric carcinoma cell line-MGC803 and regulation of GA on EphA2 and other proteinsEvery group of GA inhibited proliferation of MGC-803 cells significantly in time-dependent manner and dose-dependent manner and the highest inhibition rate reached (77.69±0.91) %; IC50 was 558.94nmol/L after 48h of GA implication. Every group of GA blocked MGC-803 cell cycle at S-phase and induced apoptosis higher significantly (P<0.01); Giemsa staining showed apoptosis morphologic change after GA: concentration and dark staining of cytoplasm, karyorrhexis and apoptotic bodies; Every group of GA inhibited the expression of EphA2 and Survivin proteins at the same time increased Caspase-3 expression (P<0.01).3 Effect of RNAi silencing EphA2 on proliferation and apoptosis of human gastric carcinoma cell line MGC-803When the ratio of cations liposome vector to plasmid vector was 2μl to 1.0μg, the transfection efficiency was 86.4%. The time was 48h by MTT and the vector was S3 by RT-PCR and Western Blot were the best for EphA2 silence. RNAi silencing EphA2 mRNA and protein in MGC803 cells inhibited proliferation, decreased after 48h, and induced apoptosis, furthermore, increased the ratio of G0/G1-phase significantly.Conclusions:1 It's the first time that detecting combinativly of EphA2, EphrinA1 and E-cadherin protein expressions in gastricarcinoma and confirming EphA2 abnormal expression in gastricarcinoma, which involved in infiltrative depth, differentiation and limphatic metastasis. Detecting the three protein levels can provide certain reference value in evaluating the metastasis, prognosis and malignant development monitoring of gastric carcinoma.2 Over expression of EphA2 in gastric cancer may inhibit cell apoptosis and promote cell proliferation, and plays an important role in the carcinogenesis of gastric carcinoma, its mechanism may not be related to the level of mRNA, which may result from the up-regulation in translational level or the increased protein stability.3 EphA2 is specifically over expressed in gastric cancer and may be involved in angiogenesis and plays an important role in the initiation and progression in gastric carcinoma.4 We provided the first evidence that GA can inhibit the proliferation, arrest cell cycle and induce the apoptosis of gastric carcinoma MGG-803 cells. The mechanism may be related with down-regulation of EphA2 and other proteins.5 It's the first time to investigate EphA2 expression in gastricarcinoma by RNAi: silencing EphA2 could inhibited proliferation and induced apoptosis. These suggested that EphA2 may be a new target for preventing gastric carcinogenesis and treating gastric carcinoma.