| Objective: Breast cancer is one of the malignant tumors which threatened the health of women. According to statistics, there are about 1200,000~1400,000 new breast cancer cases every year all over the world, and 500,000 of them died from it. Studies showed that, carcinogenesis and development of breast cancer had multiple steps and was the results of the function of multiple genes. With the increasing of the incidence of breast cancer, it becomes one hot issue in the oncology field to study the susceptibility gene and correlative gene and its product. As a negative regulation factor of cell cycle, the expression of 14-3-3σin human breast carcinoma cell was decreased, and it may be an important reason for the carcinogenesis. It had verified that p53 was the most important regulating factor of 14-3-3σ. p73αwas a shearing variant of p73. In vitro, it had verified that the expression of p73αgene increased to replace p53 to up-regulated the downstream factor 14-3-3σto exert the function of restraining the cell proliferation, when p53 gene deletion or mutation. However, the internal empirical studies about the inhibition of tumor growth of p73αand 14-3-3σhave not been carried out. In this study, we used cell exogenous gene transfection and breast cancer transplanted pattern of mouse of immunodeficiency to investigate the function of p73αand 14-3-3σto restrain the growth of breast cancer cells in vivo, and further to confirm the functional mechanism of 14-3-3σ, and the regulative function of p73αto 14-3-3σ. We anticipated to provide a new molecular biological index and target to the early discovery and targeted therapy of breast cancer.Methods:1 Cell culture and gene transfection: p53-mutate cell line MDA-MB-231 cultured by DMEM culture fluid, which contains 10% fetal bovine serum. When MDA-MB-231 mixed together about 70%, we imported pcDNA3-p53 plasmid, pcDNA3-p73αplasmid and pcDNA3-14-3-3σplasmid into the p53-mutate cell line MDA-MB-231 with lipofectamine-mediated. Free pcDNA3 plasmid was used as control.2 Breast cancer transplanted model in nude mice: We selected 3-week-old female nude mice 12, and bred in the environment of SPF. With the former group, each 2. In each group,'a'mouse was marked;'b'mouse was unmarked. After breeding for two days, we inoculated MDA-MB-231 2μg, p53 2μg, p73α2μg, 14-3-3σ2μg, p53+14-3-3σ2μg, p73α+14-3-3σ2μg(about 4×106 cells)into athymic mouse's mammary fat pad. 48 h after inoculation, we imported the interference of ADM in therapeutic dose into b mice. Tumor volumes were measured and recorded three times a week. At the end of 8 weeks, the animals were sacrificed and mammary gland samples were obtained, fixed, paraffin imbedded, chipped 4μm, HE dyeing, observed by light microscope.Results:1 p53-mutate cell line MDA-MB-231 cells grew well.2 Cell exogenous gene transfected succeed. We extracted the total RNA to detect the expression of p53, p73αand 14-3-3σat the level of transcription by RT- PCR. The result showed that, (1) p53 and p73αcan induce the expression of 14-3-3σat the transcriptional level, and 14-3-3σcan be counterproductive in p53. (2) p53 may be involved in the adjustment of p73αto 14-3-3σ.3 Tumors which were confirmed as breast cancer by pathology grew in the 14-3-3σtransfected group and the control group, 4 or 6 weeks postinoculation. No tumor grew in the p53 or p73αtransfected group, co-transfected p53/14-3-3σgroup and co-transfected p73α/14-3-3σgroup. No tumor grew in the group whose athymic mouse was injected by ADM through vena caudalis.Conclusions:1 The negative regulation factor of cell cycle 14-3-3σhas both activity and inactivity states. The activity state can inhibit the growth of tumor, while the inactivity state can not.2 p53 and p73αare both major regulation genes of 14-3-3σupstream. They can up-regulate the expression of 14-3-3σand inhibit the growth of tumor by activating it when the cellular DNA was injuried.3 As anti-oncogene, both p53 and p73αcan inhibit the growth of breast tumor cells of athymic mouse, and the concrete mechanism is to be studied.
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