Mechanisms Of Y-box Binding Protein-1C-terminal Domain In Regulating Angiogenesis

Objective: In recent years, the incidence of breast cancer is on the rise, the disease rate in female malignant tumor, poses a serious threat to the world women’s health. Angiogenesis is a key link in the process of tumor proliferation, invasion, metastasis, antiangiogenic therapy is widely used in clinical, anti-angiogenesis drugs with each passing day. Y-box binding protein-1, an important transcription factor in vivo, plays an important role on m RNA translation, DNA repair and cell proliferation and adjust in the process, and it is related to the tumor proliferation and drug resistance in lung cancer, colon cancer, gastric cancer. Studies have shown that it can adjust the proliferation of vascular endothelial cells, but the regulation mechanism of tumor angiogenesis needs further research. Using GFP and GFP-YB-1 CTD adenovirus infected cells to explore the mechanism of the cold shock protein YB-1 C-terminal domain regulating breast cancer angiogenesis, in order to provide theoretical basis for anti-tumor angiogenesis targeted therapy.Methods: Using GFP and GFP-YB-1 CTD adenovirus infected cells, then MTS method was used to detect the cell proliferation. Western blot was adopted to detect expression of cell cycle regulated protein p21 and cyclin B1, and to test m RNA level by real time PCR. Tran membrane migration experiment was used to evaluate effect of GFP-YB-1 CTD on endothelial cell EA. hy926 migration ability. Testing GFP and GFP-YB-1 CTD regulate EA.hy926 endothelial vascular ring formation and GFP-YB-1 CTD inhibit breast cancer cell secretion of promote angiogenesis factor by vascular endothelial tube formation experiments.Cytoskeleton staining was used to describe the distribution of cytoskeleton in endothelial cells EA. hy926. All the data was described with( x ± s),SPSS16.0 statistical software was used to deal with data, mean compared by t test and single factor analysis of variance(One-way ANOVA), with P < 0.05 for the difference was statistically significant.Results: According to the results of MTS method to detect cell proliferation of endothelial cells EA. hy926 and breast cancer cells SK-BR-3 after being transfected with GFP-YB-1CTD 48 hours,the proliferation rate was low compared with GFP control group(P < 0.05).Western blot analysis showed that GFP-YB-1 CTD can promote p21 protein expression in endothelial cells EA. hy926, downregulate the expression of cyclin B1 protein, on the contrary, compared with the GFP control group. Real time-PCR results indicate YB-1 CTD promote the expression of p21, and inhibit the expression of cyclin B1 on the m RNA level.Transmembrane migration experiment showed that transmembrane migration on the number of cells in GFP-YB-1 CTD group was less than GFP group. Cytoskeleton staining was observed that the GFP group EA. hy926 endothelial cells contain a lot of bulk stress fiber fascicles parallel, along the long axis of the cell. There was no obvious fiber bundle in GFP-YB-1 CTD group, depolymerization of actin filaments and diffuse distribution in cytoplasm. Endothelial tube formation experiments showed that GFP group endothelial cyclization number, vascular ring complete, relatively evenly distributed in the surface of Metrigel glue, extend the incubation time loop stability, not easy to break according to the results under microscope.Vascular endothelial cell line EA.hy962 in GFP group showed connecting and complete vascular ring. However, these vascular structure were changed to incomplete after transfected with GFP-YB-1 CTD adenovirus. ELISA was used to detect SK-BR-3 cells conditioned medium of VEGF levels, according to the results compared with GFP controls, GFP-YB- 1 CTD group of conditions medium low levels of VEGF, SK-BR- 3cells conditioned medium to stimulate blood vessels under the condition of tube formation,according to the results of GFP group endothelial cyclization quantity, blood vessels, integrity,a long circumference, extend the incubation time, loop stability, GFP- YB- 1CTD group of endothelial almost no ring or a ring formation, cyclization, incomplete and instability.Conclusion: YB-1 C-terminal domain can inhibit the cell proliferation, cell migration,vascular endothelial tube formation and the secretion of angiogenesis factors. Our results showed YB-1 CTD could be as a new target for treatment for breast cancer.