Study On The Inhibition Of Antisense Oligodeoxynucleotides Transfection On Expressions Of Paxillin In Esophageal Carcinoma Ec9706 Cells

Esophageal carcinoma is one of the common malignant tumors. China is one of the countries with high incidence and mortality of esophageal. Henan is one of the provinces with high incidence of esophageal cancer in China. The present therapies for moderately or advanced esophageal cancer are not exactly curative and effective and have some limitations. Searching new and effective treament methods of esophageal cancer has become a research hotspot in recent years.Paxillin is an important cell adhesion factor which found in recent years. It is a substrate of tumorigenicity tyrosine kinase, which attached with integrin to form the key parts for extracellular matrix adhesion and regulate local mobile and spread of cell functions, so as to improve the ability of tumor cells to metastasize and invade. As a kind of signal adaptor protein signal, paxillin plays important roles in signal transduction of many cells stimulated by external stimulus. Many studies show that paxillin has the ability to take part in dynamic adjustment on focal adhesion and on some cell functions such as mobile and spread. And the invasion and metastasis of tumor are directly related to adhesion strength and mobility of cells, which demonstrate that paxillin plays a very important role in the invasion and metastasis of cancer cells.Antisense oligodeoxynucleotide (ASODN) can inhibit gene expression specifically, but does not affect the normal function of the other genes. It has been widely used in genetic research in recent years. But it is not reported in the literature yet that the research on the effects of inhibition on the function of paxillin factor with antisense oligodeoxynucleotide in 9706 esophageal cancer cells.This study used antisense oligodeoxynucleotide restrain high technology to observe the inhibition degree of paxillin expression in esophageal cancer cells, at different time points after EC9706 are processed with paxillin ASODN with different concentration. Then EC9706 with the best effect time point and best concentration of antisense oligodeoxynucleotide transfection were transplanted in the subcutaneous of nude mice to establish the tumor models in nude mice. Then the tumor growth in nude mice and the expression of paxillin in tumor models were observed to provide experimental evidence for clinical targeted therapy of esophageal cancer.Materials and methods1. EC9706 cell line was presented by the Institute of Oncology, Chinese Academy of Medical Sciences.2. EC9706 esophageal cancer cell group:the cells with logarithmic growth were divided into ASODN experimental group with different final concentration, effect time points, N-ODN group and normal control group. In each group, there were three well plates again.3. Lipofecter using liposome mediated ASODN EC9706 in paxillin transfection and cells. Transfection of paxillin ASODN and N-ODN into EC9706 mediated with lipofecter.4.15 BALB/C nude mices with SPF degree were feeded in a barrier system with certain temperature and humidity. The feed and drinking water were sterilized.5. The establishment of transplant tumor models in nude mice:The cells from the ASODN experimental group, N-ODN group and normal group were injected into the subcutaneous of left lower limbs of nude mice which were feeded in a barrier system with certain temperature and humidity.6. The expression of paxilllin protein in different groups and transplant tumors was detected with immunocytochemistry.7. The expression of paxilllin mRNA in different groups and transplant tumors was detected with technology of in situ hybridization.8. The SPSS 10.0 statistical software was used for all statistical analyses. The differences in means were analyzed by t-test (two groups) or ANVOA (more than two groups). The relationship of two variables was analyzed by correlation analysis. P values less than 0.05 were defined statistically significant.Results1. Expressions of paxillin protein decreased with the increase of concentration or time in EC9706 transfected with paxillin ASODN. At the same time with different concentrations and same concentration with different time, there were significant differences (P<0.05), the concentration of 12μmol/L paxillin ASODN effected best at 72h time point.2. Expressions of paxillin mRNA decreased with the increase of concentration or time in EC9706 transfected with paxillin ASODN. At the same time with different concentrations and same concentration with different time, there were significant differences (P<0.05), the concentration of 12μmol/L paxillin ASODN effected best at 72h time point.3. Expressions of paxillin protein decreased in nude mice with transplant tumors in experimental group, which were lower significantly than that in N-ODN group and normal group (the P values were both less than 0.05)4. Expressions of paxillin mRNA decreased in nude mice with transplant tumors in experimental group, which were lower significantly than that in N-ODN group and normal group (the P values were both less than 0.05)Conclusion1. Paxillin ASODN can inhibit the expression of paxillin mRNA and paxillin protein in EC9706 cells.2. The transfection of paxillin ASODN can inhibit the expression of paxillin mRNA and protein in EC9706 cells with transplanted tumor in nude mice. And the paxillin ASODN can inhibit the growth of transplanted tumor for EC9706 cell in nude mice. It demonstrates the feasibility to treat esophageal cancer with antisense oligodeoxynucleotide.