Effects Of CPB2 Gene Silencing On TAFI Expression And Metastatic In The Breast Cancer Cells

ObjectiveBreast cancer is one of the most frequent malignant tumor in women and serious harm to health. Metabasis could be found in earlier period, so it is difficult to control and treat effectively.Thrombin-activatable fibrinolytic inhibitor (TAFI) is a carboxypeptidase synthesized in the liver that has been shown to play an important role in fibrinolysis. It is converted to an active enzyme (TAFIa). The process is mediated by thrombin, the thrombin-thrombomodulin complex, or plasmin. TAFIa attenuates fibrinolysis by removing the carboxyl-terminal lysine residues from partially degraded fibrin that mediate positive feedback in the fibrinolytic cascade. Higher levels of TAFIa may correspond to an increased risk of thrombosis. It is involved in many kinds of diseases such as vascular disease and cancer. The chromosomal localization of the gene encoding TAFI (carboxypeptidase B2, CPB2) was determined to be 13q14.11. TAFI protein contains 423 AA (22 AA signal peptide,92 AA active peptide, and 309 AA action domain).We transfected with the siRNA targeting CPB2 into the breast cancer cell line MDA-MB-231, and then observed the effects of gene silencing on TAFI expression. Then to explore the proliferation and invasion ability in the breast cancer cell line MDA-MB-231, so we can detect the significance of TAFI in tumor. Furthermore, we can investigate the relationship of the fibrinolytic system and tumor.Methods1. The human breast cancer cell line MBA-MB-231 were cultured and passaged;2. Three specific siRNA and a nonspecific siRNA were designed. MDA-MB-231 cells in the exponential phase of growth were divided into five groups (siRNA-1, siRNA-2, siRNA-3, negative control group and blank control group), and then transfected with siRNA using lipofectamine 2000;3. RT-PCR detected TAFI mRNA expression inhibited in MDA-MB-231 cells;4. Western blot detected TAFI expression inhibited in MDA-MB-231 cells;5. We selected the siRNA-3 group to be the highest efficient siRNA through RT-PCR and WB. The invasion was analyzed by transwell assay with the siRNA-3 group;6. The proliferation was analyzed by MTT with the siRNA-3 group.Result1. Compared with negative control group and blank control group, TAFI mRNA expression in MDA-MB-231 cells of siRNA groups which measured by RT-PCR was inhibited significantly (P<0.05).2. TAFI expression in MDA-MB-231 cells of siRNA group was inhibited significantly analyzed by western blot (P<0.05), and the inhibition ratio range from 55.4% to 42.6%.3. Transwell assay showed that invasion ability was inhibited in siRNA-3 group compared with negative control group and blank control group.4. MTT assay indicated that proliferation was decreasing in siRNA-3 group.Conclusion1. Expression of TAFI was inhibited by RNAi, after transfected, TAFI mRNA and TAFI expression was decreasing significantly. Especially the inhibition effecting of siRNA-3 group was highest.2. The decreasing of TAFI could cut down the proliferation and invasion ability of MDA-MB-231 cells.3. Through decreasing the proliferation and invasion ability, TAFI expression was involve in tumor closely. TAFI will become a new idea.