Effects Of SPINK8Gene On Proliferation、Apoptosis And Migration Of Esophageal Carcinoma9706

Background and Aims:Esophageal cancer (EC) is one of the most aggressive malignant tumor in the upper digestive tract. It can be divided into esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) according to histological types with different clinico-pathologic. ESCC was associated with chronic stimulation and inflammation factors such as drinking, smoking and so on. EAC is increased in the setting of Barrett’s esophagus (BE), a premalignant lesion characterized by replacement of the normal esophageal squamous epithelium with a specialized intestinal metaplasia. Esophageal cancer greatly reduced the quality of life because of its clinical features:swallowing difficulty, food reflux, even bleeding. Recent years, although advances of surgery procedures and widely application in multimodality therapy, the overall5-year survival rate for EC is still remain poor, about14%. At present, EC is still the sixth killer overall the world.It was considered that EC was a complex disease, in which many genes take part and go through a lot of stage developments. Many studies discussed alterations of oncogene and anti-oncogene during the development of EC. Li et al extracted RNA from ESCC tissues and matched normal esophageal epithelium, then using whole genome microarrays and bioinformatics to analyze which genes may be important in the carcinogenesis of ESCC. The results showed that SPINK8gene was down-regulated in ESCC tissues compared with normal esophageal epithelium. It suggested that SPINK8gene may be a new anti-oncogene which play a key role during the development of EC.Thus, we use RT-PCR methods to get the SPINK8gene from peripheral blood of normal person, then construct recombinant vector and transfect the esophageal carcinoma9706cells. Using flow cytometer, MTT and transwell methods to study the effects of SPINK8on esophageal carcinoma9706.Mehtods:1. Total RNA were extracted from peripheral blood of normal person, reverse transcribed to cDNA. Then use PCR method to obtained SPINK8gene.2. Construct recombinant vector and induce prokaryotic expression to make sure SPINK8gene can express correctly.3. Construct eukaryotic recombinant vector and transfect EC9706cell, then select stable transfection cells.4. Use MTT colorimetric method to determine the effect of SPINK8gene on proliferation activity of EC9706cells.5. Use flow cytometry method to detect the effect of SPINK8gene on cell apoptosis of EC9706cells.6. Use Transwell migration experiment to test the effect of SPINK8gene on cell migration of EC9706.7. All statistical tests were performed with SPSS version17.0. T test was used to compare the differences between two groups. The P value for significance was set at0.05.Results:1. Results of MTT:compared with non-transfected group, the number of live cells of transfected recombinant plasmid group was decreased, and inhibition ratio was24.53%. The difference between recombinant plamid group and non-transfected group was statistically significant, P=0.013. while there was no significant difference between null plasmid group and non-transfected group, P=0.145.2. Results of flow cytometry:the apotosis rate of recombinant plamid group was significantly higher than that of non-transfected group the P value was0.014. When compared non-transfected group with null-plamid group, the difference was not statistically significant, P=0.057.3. Results of Transwell:when compared with non-transfected group, the recombinant plamid group had a lower number of cells which passed through the transwell, and the differences was statistically significant, P=0.003. While non-transfected group and null-plamid transfected group had no difference, P=0.195.Conclusion:1. SPINK8gene has a inhibitory effect on proliferation of EC9706and promotes cell apotosis. 2. SPINK8gene can reduce the ability of cell migration of EC9706.