The Inhibition Of Spink8 Gene On Proliferation And Migration Of Esophageal Carcinoma 9706

Background and Aims Esophageal cancer is the malignant tumor in esophageal epithilial tissue, there are about 20 million people around the world died of esophageal cancer each year, poses great threat to people life and health. the etiology of esophageal cancer has not yet fully understood, it has been found that the incidence of esophageal cancer is associated with genetics, nitrosamines chronic irritatioin, inflammation, trauma, and content of trace elements in drinking water, food and vegetables through multiple avenues, But the exact cause is not clear. EC9706 cells derived from esophageal carcinoma cell lines, taking EC9706 cells as the research object in vitro has the important value in exploring and understanding the biological behavior of esophageal cancer. SPINK family is a kind of serine protease inhibitor family, Kazal-like, including SPINK 1 to 12 subfamilies, a number of the family members are related to diseases and tumor, such as spink1 related to pancreatic cancer, spink2 corelated with male infertility, and spink7 has relationship with esophageal cancer. spink7 has been conformed to be a tumor-suppressing gene, which has the function of regulating cell proliferation, inducing apoptosis and ingibiting tumor migration and invasion. The previous study found that the expression of SPINK8 in adult esophageal cancer tissue was down-regulated compared with normal esophageal carcinoma tissues. The reports about the function of SPINK8 protein was rarely. spink8 and spink7 have homology, and the simulation of three-dimensional structure is similar, however, there are some differences between the amino acid sequences of the two proteins, which is speculated that the two protein may inhibit different proteases. This study will construct recombinant plasmid of sh RNA based on NCBI databse records, and transfect the recombinant plasmid into EC9706 cells mediated by lipid body, observe the expression level of spink 8 gene to discuss the affects of SPINK8 protein to EC9706 in vitro about proliferation, migration and colony forming ability. The experiment will explore whether spink 8 is a tumor suppressor gene, and discuss its mechanism to provide new theoretical mechanism for the occurrence and development of esophageal cancer and new targets for treatment.Mehtods 1. Construct p Genesil-SPINK8 recombinant by the target of spink8 m RNA sequence. 2. Transfect EC9706 cells by recombinant plasmid p Genesil-SPINK8, negative control plasmid, over express plasmid p EGFP-C1-SPINK8 and blank plasmid p EGFP-C1, named the groups as blank group, interference group, negative group, blank plasmid group and overexpression group, testing the level of m RNA and protein by RT-PCR and Western Blotting. 3. Detect the proliferation of five groups after transfection by MTT. 4. Use scratch assay to test the migration ability of transfected EC9706 cells. 5. Compare the changes of clony formation ability by single-clone formation assay. 6. Statistics: All statistical tests were performed with SPSS version 17.0. T test was used to compare the differences between two groups. The P value for significance was set at 0.05.Results 1. Constructed the recombinant plasmid p Genesil-SPINK8 successfully. When digested the recombination by restriction enzyme SalⅠ, we get a band at 400 bp; the results of sequencing confirmed with our design. 2. Transfected EC9706 cells by recombinant p Genesil-SPINK8 plasmid, the inhibition of m RNA and protein expression are significance by RT-PCR and Western Blotting. 3. The expression of m RNA and protein of SPINK8 was increased significance at p EGFP-C1-SPINK8 EC9706 cells(P<0.05). 4. MTT growth curve showed that over express SPINK8 protein could inhibit EC9706 cells proliferation, but interference its endogenous expression could increase the proliferation in vitro. 5. Scratch healing assay confirmed that over express SPINK8 protein could decrease the horizontal migration ability of EC9706 cells, but know down its endogenous expression woμld speed up cell migration velocity. 6. Clony formation assay displayed that the single-clone formation ability of the over express EC9706 group was decline, but interference group was enhanced both at number and size.Conclusion 1. spink8 gene has the inhibitory effect on proliferation of EC9706. 2. The si RNA sequence can effectively inhibit the endogenous expression of SPINK8 protein in EC9706 cells. 3. spink8 gene can reduce the ability of cell migration and clone formation ability of EC9706. 4. spink8 gene has a certain inhibitory effect on the migration of EC9706 in vitro.