Study On The Expression Of EphrinA2 And EphA7 In Esophageal Cancer

Esophageal cancer is one of the most common tumors in China, accounting for half of global number cases. The Taihang Mountain region is where esophageal cancer frequently occurs, including the Linxian of Henan, Cixian of Hebei, Yangcheng of Shanxi, and so on. Environmental factors and genetic factors involved in the evolution of esophageal cancer. The differernces of the regional distribution of esophageal cancer and the family gathering phenomenon in high occurrence regions suggest that genetic factor play an important role in the occurrence of disease.Eph family is the largest known receptor tyrosine kinases family.Eph and Ephrin extensively participate in the starting and regulation of a variety of biological events. Eph and Ephrin are high expression in a variety of tumors, and show the characteristi cs of promoting tumor progression. EphrinA2 is an important ligand of the EphA fa-mily, but few studies with tumors, mainly in breast cancer cells and hepatocellular ca-rcinoma. EphA7 is a receptor of EphrinA2, and closely related to tumor. The express-ion of EphA7 in liver cancer, colon cancer, stomach and other digestive tumors have been reported.ObjectiveThis study assays EphrinA2 and EphA7 mRNA and protein expression levels in esophageal cancer organization, and analysis the relationship between expression level with the clinical pathology parameters in patients, and study the influence of EphrinA2 to esophageal cell lines.Material and Methods1. Subjects, samples and clinical data92 subjects were in patients diagnosed as esophageal cancer waiting for surgery in Linxian Tumor Hospital from 2007 to 2008. They were willing to take part in the study. The patients did not receiving any forms of chemotherapy and radiotherapy treatments. The general information and clinical information of the subjects were collected.2. Expression of EphrinA2 and EphA7 mRNA in samples To determine the mRNA expression levels of EphrinA2 and EphA7 in esophageal cancer tissues and paired normal esophageal tissues by real-time PCR, regardingβ-actin as the reference gene.3. Detection of EphrinA2 and EphA7 protein in samplesTo detect the expression levels of EphrinA2 and EphA7 protein in cancer tissues and the corresponding normal tissues by western blot and immunohistochemistry. All samples were observed at 200×magnification under the three most colorful focuses, regarding cytoplasm stained brown was positive, and calculated the numbers of cell expressing dyeing positive protein. The percentage of cells express dyeing positive protein was estimated by dividing the numbers of positive immunostaining cells by the total numbers of cells per high-power focus.4. Detection expression and growth of EphrinA2 in cellTo detect the expression of EphrinA2 in eight esophageal cancer cells by Western blot. To detect the growth of KYSE-180 cells by enzyme-linked immunosorbent detector, draw spectrophotometry the growth curve.5. Data analysisThe data was analysis through the statistical analysis software SPSS 12.0, using t test comparison EphrinA2 and EphA7 mRNA expression levels in 92 cases of esophageal cancer organizations and their paired normal esophageal tissues, using-χ2 test analysis the relationship between EphrinA2 and EphA7 mRNA expression levels with clinical parameters. The level of significance was 0.05.Results1. Real time PCR results in esophagus tissues1.1 Expression levels of EphrinA2 and EphA7 mRNA in esophagus cancer tissues and paired normal esophagus tissuesFluorescence quantitative PCR results showed that in 92 esophageal samples EphrinA2 and EphA7 mRNA levels over expression respectively in 59.8% (55/92), 73.9%(68/92) of esophageal cancer tissues, compared to normal esophageal tissues. Matching t-test shows that the EphrinA2 and EphA7 mRNA expression levels in cancerous tissues and the paired normal tissues were significant difference(t=5.695, P<0.05; t=7.179, P<0.05), the expression levels in cancerous level tissues were significantly higher than the paired normal tissues. Pearson's related analysis shows that EphrinA2 and EphA7 gene mRNA expression levels in esophageal tissues are highly positively related(r= 0.283, P<0.05)1.2. The relationship between EphrinA2 and EphA7 gene mRNA expression levels and the clinical pathology factors of esophageal cancerEphrinA2 mRNA expression level in different pathological types have statistically significant (χ2=6.463, P<0.05). EphrinA2 mRNA expression level in lymph node metastasis group significantly higher than without lymph node metastasis group (χ2=6.800, P<0.05). EphrinA2 mRNA expression level in tumor family history higher than without tumor family history group (χ2=6.049, P<0.05)The mRNA expression level of EphA7 in different age groups are statistically significant difference (χ2=6.506, P<0.05). EphA7 mRNA expression level in lymph node metastasis group significantly higher than without lymph node metastasis group (χ2=11.272, P<0.05).2. Immunohistochemical staining and Western blot results in esophagus tissues2.1 Expression levels of EphrinA2 and EphA7 protein in esophagus cancer tissues and paired normal esophagus tissuesImmunohistochemical stains and Western blot results:the expression levels of EphrinA2 and EphA7 can be seen in patients with esophageal cancer higher in esophageal cancer tissues than paired normal esophageal tissues.2.2 The relationship between EphrinA2 and EphA7 gene protein expression levels and the clinical pathology factorsEphrinA2 protein expression level in tumor family history group significantly higher than without tumor family history group (χ2=4.304, P<0.05). EphrinA2 protein expression level in lymph node metastasis group significantly higher than without lymph node metastasis group (χ2=4.304, P<0.05). In lymph node metastasis group, EphA7 protein expression level significantly higher than without lymph node metastasis group (χ2=4.800, P<0.05).3. To detect the EphrinA2 expression lever in Human esophageal squamous cancer cell lines and growth affection of EphrinA2 in KYSE-180 cell linesWestern blot results showed that the expression lever of EphrinA2 in KYSE-180 cell line is minimum. Determined by MTT colorimetric method shows:KYSE-180/ EphrinA2H, KYSE 180/EphrinA2L and KYSE-180v cell spectrophotometry in every day has KYSE-180 EphrinA2 H> KYSE/180 EphrinA2 L>KYSE-180 v.Conclusion1. EphrinA2 and EphA7 mRNA and protein expression levels in esophageal cancer organizations were significantly higher than esophageal normal organizations. The expression levels of EphrinA2 and EphA7 mRNA were significantly positive correlated, and both maybe interaction in the process of tumorigenesis and development.2. EphrinA2 mRNA and protein expression levels closely related lymph node metastasis and family history. EphA7 mRNA and protein expression levels closely related to lymph node transfer.3. In cell experiments suggests EphrinA2 gene can promote esophageal cancer cell proliferation.4. EphrinA2 and EphA7 genes can be used as judge the related gene transfer esophageal patients, EphrinA2 genes may be a new target of gene therapy.