A Research On What Role Does 14-3-3θ Play In Breast Cancer’s Biological Behavior And Potential Mechanism

BackgroundThe global incidence of breast cancer increased in recent years, breast cancer has become one of the most common malignant tumors to women, and it causes serious harm to the health of women. treatment of breast cancer has a rapid development, the survival of breast cancer has been significantly prolonged. But due to breast cancer cell migration and resistance of drug, eventually lead to breast cancer recurrence and metastasis. So to find an effective molecular target for breast cancer prognostic and diagnosis will provide a new strategy for the treatment of breast cancer, it has important clinical significance.14-3-3 protein family is widely expressed in eukaryotic cells, and it is s a kind of highly conservative multifunctional acid dimers protein.14-3-3 protein family, there are seven subtypes encoded by different genes(beta, gamma, epsilon, sigma, eta, theta and zeta) in 14-3-3 protein family. They play important roles in regulation of intracellular signal transduction, metabolism, cell cycle and apoptosis. Existing researches show that only 14-3-3sigma can reduce output of NF-kappa B, so as to play a role of inhibition of tumor. The other subtypes are linked with cell invasion, proliferation, migration indifferent tumors. 14-3-3theta subtype high expressed in meningioma, and the more 14-3-3theta expressed, the higher degree of meningioma there are. The 14-3-3theta is associated with the deterioration of meningioma. A study suggests that the expression of 14-3-3theta is linked with lymph node metastasis of breast cancer, so we speculated that the 14-3-3theta may be a potential forecast factor of the invasion and metastasis of breast cancer. But how 14-3-3 theta impacts on the biological behavior of breast cancer and potential molecular mechanism is still not clear. What is the effect of 14-3-3theta on breast cancer cell proliferation, cycle and apoptosis? Is 14-3-3theta associated with the prognosis of breast cancer? Can 14-3-3theta be a new therapeutic targets for breast cancer? With these questions, firstly we build breast cancer cell lines which can overexpress 14-3-3theta. And then to find out the effect 14-3-3theta on breast cancer cell biology behavior and its mechanism.Methods and results1. Build and screen breast cancer cell lines which can overexpress 14-3-3theta: we ordered the p ENTR 3c DUAL carrier and then put 14-3-3theta gene into plasmid vector pc DNA4.0. After transfected 293 T cells, we indentified it by western blot. We used the pc DNA4.0-14-3-3θ to transfect breast cancer line AU-565、MCF-7 and BT-474, after screened by zeocin, we got the breast cancer cell lines which can overexpress 14-3-3theta.2. Research about he effect of 14-3-3theta on breast cancer cell biology behavior:(1) western blot experiments tested the expression level of 14-3-3theta in five breast cancer cell lines.(2) we determined MCF-7 and AU-565 cell proliferation activity by MTT experiment after increased the expressin of 14-3-3theta;(3) Plate clone formation assay determined MCF-7 and AU-565 ability of clone formation after increased the expressin of 14-3-3theta;(4) We observed the apoptosis of MCF 7 and AU-565 by flow cytomet after increased the expressin of 14-3-3theta(5) Scratch experiment detected MCF-7 and AU-565 migration ability after increased the expressin of 14-3-3theta;(6) We observed the growth of tumor in nude mice, and then do IHC with the tumor tissues. Results: the difference expression of 14-3-3theta in five breast cancer lines may be correlated with the malignant degree of breast cancer. Up-regulation of 14-3-3theta can promote breast cell proliferation, migration, clone formation ability, reduce breast cancer cells apoptosis and promote the growth of tumor in nude mouse. Above all, 14-3-3theta can promote the malignant biological behavior of breast cancer. Up-regulation of14-3-3theta in MCF-7 and AU-565 can decrease sensitivity of breast cancer cell to doxorubicin, tamoxifen, and herceptin.3. A study about the mechanism of 14-3-3theta decreased sensitivity of breast cancer cell to doxorubicin, tamoxifen, and herceptin(1) We detected the amount of m TOR、p-m TOR、Akt、p-Akt in AU-565 and MCF-7 by western blot, and we detected the amount of the Bcl-2, Bax and PARP after we added herceptin to AU-565 for 72 h.(2) We observed the growth of tumor in nude mice which were injected by herceptin, and then do IHC.Results: 14-3-3theta reduced breast cancer cell sensitivity to adriamycin, tamoxifen, it may be linked with increased activation of Akt and m TOR. 14-3-3theta reduced breast cancer cell sensitivity to herceptin, it may be associated with increased activation of Akt and m TOR, the more Bcl-2 and PARP, the fewer BAX.Conclusion:1. The expression of 14-3-3 theta is difference in five breast cancer lines may be linked with the malignant degree of breast cancer. 2. 14-3-3theta can romote the malignant biological behavior of breast cancer cells. 3. Up-regulation of 14-3-3theta can reduce breast cancer cells sensitivity to adriamycin, tamoxifen and Herceptin by increased the activation Akt-m TOR pathway...