ANXA2 Caco2 On Biological Behavior Of Cells

Background and Objective:Colorectal cancer is a common clinical gastrointestinal disease that terribly endangers human life wordwide. Recently, the incidence of the disease tends to rise year by year because of the change of people’s life style and the irrationality of dietary pattern. At present, the surgery is the most primary and effective treatment of the disease, but the survival rate in five years doesn’t enhance significantly all the time. The development of molecular biology brings a new hope to people from tumor gene therapy. The key step of gene therapy is the establishment of target gene. Like other cancers, the oncogenesis of colorectal carcinoma is also a complicated pathological process involved with many factors, genes and stages. As the member of annexin family, The ANXA2protein is involved with a serial of vital biological activities including membrane formation, membrane transportation, exocytosis, endocytosis, cell proliferation, signal transduction, cell differentiation and apoptosis. Recently, study data showed that the up-regulation of ANXA2protein level in most cancer tissues is closely related to oncogenesis, cancer development, invasion and metastasis. Knockout as a new method of molecular biology is dependent on the homologous recombination and technology of embryonic stem cell, because of specificity and effectivety, now it has emerged as a powerful tool for gene function research, and provided with great applicable foreground in the tumor gene therapy.In this study, we employ Knockout to delete ANXA2expression and establish ANXA2-/-caco2cell line, through the changes of proliferation, migration and apoptosis to study the effects of ANXA2to caco2’s biological behavior. In experimental condition without ANXA2expression, use PCR and western blot to detect the expression level of related genes S100A10, FAK, P-actin, tPA to analyze the role of them to proliferation, migration and apoptosis. To explore the relationship and some mechanism between ANXA2and S100A10, FAK, P-actin, tPA and try to find out the relevant signaling pathways involved in colorectal cancer development process, so as to further explore the ANXA2’s role in colon cancer’s cause and development, as well as its regulatory role in the process of tumor development. Methods:1. Western blot method is applied to confirm no ANXA2expression in caco2cell lines (ANXA2-/-caco2).2. MTT is applied to test cell activity of caco2, to analyze the ANXA2’s influence on caco2cell proliferation at different time pionts (24h,48h,72h,96h,120h).3. Wound healing assay to confirm the influence of ANXA2to caco2’s motility.4. Hoechst33342staining, MitoView TM633staining and flow cytometry assay were performed to measure the effects of ANXA2on cell apoptosis.5. The expression level of S100A10, FAK, β-actin, tPA were analysised by RT-PCR and western blot to study the effects of depletion of ANXA2on them.Results:1. Western blot was further confirmed the ANXA2-/-caco2cell line constructed successfully.2. MTT revealed that the ability of proliferation was inhibited markedly when the ANXA2deleted (**P<0.01).3. Results of Wound healing assay show that the ability of migration decreased significantly in ANXA2-/-caco2cells.48h later appear clearly inhibited (*P<0.05).4. Hoechst33342staining, MitoViewTM633staining, flow cytometry assay revealed that the the apotosis of caco2no changed when the ANXA2depletion.5. RT-PCR, western results showed that after knockout ANXA2, S100A10, tPA expression level decreased significantly (**P<0.01), and the expression level of β-actin, FAK is not obviously changed (P>0.05).Conclusion:ANXA2is closely related to the caco2cell proliferation and migration, after ANXA2knockout, its proliferation, migration ability is decreased, but do not significantly affect their apoptosis. The expression levels of S100A10, tPA significantly change when ANXA2deleted (**P<0.01), Oppsite, FAK and P-actin expression it makes no difference. Under the condition of target gene knockout, the results of the present study from a more objective and accurate angle further support ANXA2on colon cancer development-related characteristics of an important regulatory role, the S100A10, tPA, FAK, β-actin are involved in proliferation, migration, and metastasis of caco2cells which are detected by RT-PCR and western blot. The interaction among S100A10, tPA, FAK, β-actin and ANXA2are co-regulated the development of colon, Revealed that ANXA2might be a potential target for gastric carcinoma gene therapy or drug therapy.