Effects Of Recombinant PcDNA3.1-Annexin A1 Plasmid On Migration And Invasion Of Colon Cancer Cell Line SW480

The annexin superfamily consists of 13 calcium or calcium and phospholipid binding proteins with a significant degree of biological and structural homology (40-60%). Annexin A1 (ANXA1), the first characterized member of the annexin superfamily, is a member of multifunctional protein shown to regulate a variety of cellular processes in epithelial cells including endocytosis/exocytosis, proliferation, differentiation, as well as eicosanoid production. Recently, Annexin Al has been reported that it is associated with the development of metastasis in some invasive malignancies suggesting a role for this protein in regulating epithelial cell migration/invasion. However, mechanisms by which Annexin A1 regulates epithelial eel lmigration/invasion are not understood. In our study, we constructed the eukaryotic expression vector of pcDN A3.1-Annexin A1 and transfected it stably into the SW480 cell lines, to study the effects of Annexin A1 on migratory and invasive ability of colon cancer cell line, provides a fundamental tool to further study the role of Annexin A1 gene in invasion and metastasis of human colorectal carcinoma.Part 1. Construction of eukaryotic expression vector of human Annexin A1 GeneObjective:To construct the eukaryotic expression vector of pcDNA3.1-Annexin A1, provides a fundamental tool to further study the role of Annexin A1 gene in invasion and metastasis of human colorectal carcinoma.Methods:Fresh normal human placenta total RNA were extracted and the Annexin A1 gene was amplified by RT-PCR and construct the eukaryotic expression vector of pcDN A3.1-Annexin A1.Identification of the recombinant plasmid pcDNA3.1-Annexin A1 by PCR,restriction enzyme analysis and sequencing.Results:Restriction enzyme analysis and sequencing result showed that the sequence of this gene fragment was identical with Annexin A1 gene sequence reported in GenBank.Conclusion:The recombinant plasmid pcDNA3.1-Annexin Al was successfully constructed.Part 2. The biological effects of recombinate vector in transfected colorectal carcinoma cell line SW480Objective:pcDNA3.1-Annexin A1 was transfected into colorectal carcinoma cell line SW480,to observed and study the biological behaviors of SW480 before and after transfection in vitro.Methods:Two different plasmids including a recombinate pcDNA3.1-Annexin A1 and an empty pcDNA3.1 were reseparately transfered into colorectal carcinoma cell line SW480 cultured in vitro by liposome-mediated gene transferred method.After screening with G418,the stable transfectants were obtained.Realtime PCR and Western blot methods were used to analyze the expression of Annexin A1 mRNA and protein in SW480 cells before and after transfection to confirm whether the recombinant vector DNA integrated with the genomic DNA of SW480 cells. Wound-healing experiment and Transwell invasion assays were used in vitro to study the effects of Annexin A1 expression on the migratory movement and invasion of SW480 cells.Results:The results of real time PCR and Western blot showed that the Annexin A1 expression was higher in cells transfected with pcDNA3.1-Annexin A1 than in those un-transfected or empty vector-transfected cells and there is no significant difference between the latter two groups. Transfected with Annexin A1 eukaryotic expression vector could significantly increase migratory and invasive ability of SW480 cells compared with the untransfected cells.The migratory rate of SW480 cells in pcDNA3.1-Annexin A1 group was significantly higher than those in un-transfected cells or those transfected with empty vectors ([0.415±0.0022] vs [0.267±0.002] and [0.271±0.002], P<0.05), and there was no significant difference between the latter two groups.The migrating SW480 cells in pcDNA3.1-Annexin A1 group was significantly more than those in the other two groups ([221.75±12.07] vs [162.8±12.07] and [164.25±9.5], P<0.05).Conclusion:The eukaryotic vector was transfected successfully to SW480 cells with liposome, which integrated with SW480 cell genome and markedly enhanced the expression of SW480 mRNA and protein in SW480cells; overexpression of Annexin Al eukatyotic expression vector can incerease migratory and invasive ability of SW480 cell line.