Empirical Research Of The Expression And Correlation Of P73α And 14-3-3σ In Breast Cancer

Breast cancer is one malignant tumor 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. Recent years, the incidence of breast cancer in China had increased by 3% every year. Specialists anticipate that breast cancer would be the most common malignant tumor in China 20 years later. Statistics also showed that age of breast cancer onset in China was 10 years earlier than that of western countries. The peak age of onset is 40～45, and more and more younger, which means that breast cancer would threaten the life of thousands of women, the intact of thousands of families and the normal work of millions of labour force. Therefore, to investigate and know the correlative factors of carcino genesis and development of breast cancer would significantly meaningful to decrease the incidence of breast cancer and to enhance the survival rate of the breast cancer patients.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. P73 gene was a new gene found by Kaghad in 1997 by chance, and its exon had considerable correlation with p53 gene, and thus it was named p73 gene according to the molecular mass of its codogenic protein. P73 gene was located in chromatosome 1p36.2-3, and this domain was an absent hot spot in the cells of neuroblastoma. It was also absent in some other tumors, such as breast cancer, colon carcinoma and melanoma. Many shearing variants were generated after the genetic transcription of p73 gene, which was different from p53 gene. As yet, at least 6 shearing variants of p73 were found, they are p73α, p73β, p73γ, p73δ, p73εand p73ζ, and p73αwas expressed in the form of total length.14-3-3σwas a member of 14-3-3 family which includingβ,ε,γ,η,τ,ζandσ, and it was the only one who could be induced by DNA damage. 14-3-3σwas a negative regulation factor of cell cycle, whose activation could make cell cycle arrest in G2 stage, then suppress the growth of cells. It was found that the expression of 14-3-3σin human breast carcinoma cell had a decreasing trends, then it could be a specific mark of human breast epithelium, and the absence of the carcino-suppression function of 14-3-3σmay be an important reason for the carcinogenesis. Therefore, to study the regulation of 14-3-3σon the gene level maybe a new breakthrough to the study of carcinogenesis and development of breast cancer. It had vertified that p53 was the most important regulating factor of 14-3-3σ, and an isomeride of p63△Np63 can also regalate the activity of 14-3-3σ. Concerning the relation between p73 and the activity of 14-3-3σ, and their expression in the carcino genesis and development of breast cancer hadn't been lucubrated up to now.We detected the expression of carcino-suppression gene p73αand a negative regulation factor of cell cycle 14-3-3σin the normal breast tissues, tissues besides tumor and tumor tissues and the interaction between them on the transcriptional and protein level by using Reverse transcription- polymerase chain reaction and Streptavidin peroxdase conjugated method; We also investigated the regulative function of p73αto 14-3-3σin the p53 mutated human breast cancer cell line MDA-MB-436 by using Reverse transcription-polymerase chain reaction, Immunoprecipitation, western blot and gene transfection methods; Studed the regulative function of 14-3-3σto the genetic transcriptive activity of p73αby using luciferase report agent analysis, Reverse transcription-polymerase chain reaction, Western-blot and colony forming experiment; Study the influence of 14-3-3σto the gene stability of p73αby using gene transfection, Reverse transcription-polymerase chain reaction, Western blot, and Cycloheximide protein restrain analysis; 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 tumor cells, and further to confirm the functional mechanism of 14-3-3σ, and the regulative function of p53 and p73 to 14-3-3σ; Together with the retrospective analysis of the correlation of the expression of p73αand 14-3-3σin the breast cancer tissue and their clinicobiological behaviour, we anticipated to provede a new molecular biological index and target to the early discovery and targeted therapy of breast cancer. And illuminate the regulative function of p73αto 14-3-3σfor the first time both domestic e and overseas.The research contents and results are as follows:PartⅠStudy of the expression of p73αand 14-3-3σin the benign and malignant tissues of breast and the relationship between their expression and the clinical biological behaviourObjective: Detect the expression of p73αwhich was a new member of p53 family and the negative regulative factor of cell cycle 14-3-3σin the benign and malignant tissues of breast, then investigate the interaction of the two gene in the carcinogenesis and development of breast cancer, and the relationship between their expression and the clinical biological behavior of breast cancer.Methods: Detect the expression and interaction of anti-oncogene p73αand the negative regulation factor of cell cycle 14-3-3σin the normal breast tissues, tissues besides tumor and tumor tissues on both transcriptional and protein level by using reverse transcription-polymerase chain reaction and streptavidin peroxdase conjugated method. And retrospectively analyze the relationship between the expression of p73α, 14-3-3σand the clinical biological behavior including tumor size, TNM stages, pathological types, histology grades, metastasis of axillary lymph nodes, haemal tube tumor embolus, ER/PR and HER-2.Results: 1 Expression of p73αin the normal breast tissues, tissues besides tumor and tumor tissues1.1 Expression of p73αon the mRNA levelP73αwas positively expressed in 25 of 33 normal breast tissue (76%); 22 of 33 were positively expressed in the tissues besides tumor (67%), and 20 of 33 were positively expressed in the tumor tissues (61%). The rectificational value of p73α(p73/β-action)in the normal breast tissues, tissues besides tumor and tumor tissues were respectively 0.8378±0.5921,0.9199±0.8441 and 0.6150±0.3756, and no significant difference was found among the three groups (F=1.139,p=0.326).1.2 Expression of p73αon the protein levelP73αprotein was positively expressed in 25 of 33 normal breast tissue (75.7%); 30 of 33 were positively expressed in the tissues besides tumor (90.9%), and 10 of 33 were positively expressed in the tumor tissues (30.3%). Significant difference was found between the tumor tissues and tissues besides tumor or normal tissues (χ2=17.860, p=0.000;χ2=8.726, p=0.003), and no significant difference was found between the tissues besides tumor and the normal breast tissues (χ2=3.385, p=0.066).2 Expression of 14-3-3σin the normal breast tissues, tissues besides tumor and tumor tissues2.1 Expression of 14-3-3σon the mRNA level14-3-3σwas expressed in all the 33 normal breast tissues; 32 of 33 were positively expressed in the tissues besides tumor (97%), and were positively expressed in all the 33 tumor tissues (100%). The rectificational value of 14-3-3σ(14-3-3σ/β-action)in the normal breast tissues, tissues besides tumor and tumor tissues were respectively 2.8231±1.9734,2.1950±1.8636 and 1.4680±0.8401, and no significant difference was found between the tumor tissues and tissues besides tumor, and between the tissues besides tumor and the normal breast tissues (p=0.075, p=0.123); The expression of 14-3-3σin the tumor tissues were much lower than that of the normal breast tissues, and significant difference was found (F=5.640, p=0.005).2.2 Expression of 14-3-3σon protein level14-3-3σprotein was strongly positively expressed in 18 of 33 normal breast tissue (54.5%); 11 of 33 were strongly positively expressed in the tissues besides tumor (33.3%), and 6 of 33 were strongly positively expressed in the tumor tissues (18.2%).The expression level of 14-3-3σprotein was negatively correlative with the cellular proliferation (r=-3.093, p=0.003); And significant differences were found between the normal breast tissues and tumor tissues(χ2=14.029, p=0.000), between tissues besides tumor and tumor tissues (χ2=6.344, p=0.012), but no significant difference was found between the normal breast tissues and tissues besides tumor (χ2=2.752, p=0.097).3 Relationships between the expression of P73αprotein and the clinical parameters16.7% (4/24) were P73αpositive in the haemal tube tumor embolus group; and 40.4% (21/52) were p73αpositive in the no haemal tube tumor embolus group, and significant difference was found (χ2=4.185, p=0.034). In the patients of ER+PR+, the positive rate of p73αwas 72.7% (16/22); and in the patients of ER-PR-, the positive rate was 33.3% (8/24); and only one was positively expressed in the ER+PR-/ ER-PR+ group, the rate was 3.3% (1/30). Significant differences were found in every two groups (χ2=27.692, p=0.007). Relationships between the protein expression of p73αand the other clinical parameters: no significant correlation were found between the p73αexpression and clinical stages (χ2=0.217, p=0.887),metastasis of axillary nodes (χ2=0.007, p=0.564),numbers of metastasis of axillary nodes (χ2=0.002, p=0.579),tumor size (χ2=0.953, p=0.329),pathological types (χ2=1.907, p=0.167) histology grades (χ2=0.02, p=0.888) and the over-expression of HER-2 (χ2=1.931, p=0.126).4 Relationships between the protein expression of 14-3-3σand the clinical parametersPositive rate of 14-3-3σprotein expression decreased along with the increase of the histology grades, and negative correlativity was found between them (γ=-0.369,χ2=6.133, p=0.013); In the group whose HER-2 was hyper-expressed (more than ++), the strong positive rate was 16.8% (8/48), and in the group whose HER-2 was hypo-expressed, the rate was 44.4% (12/28), and significant difference was found (χ2=6.173,p=0.014); No significant differences were found between the expression of 14-3-3σand pathological types (χ2=1.462, p=0.227), metastasis of axillary nodes (χ2=1.373, p=0.179), number of the metastasis of axillary nodes (χ2=0.468, p=0.349),haemal tube tumor embolus or not (χ2=1.684, p=0.154) , tumor size (χ2=1.317, p=0.251), clinical stages (χ2=0.079, p=0.779) and ER/PR (χ2=0.044, p=0.834).5 Relationship between 14-3-3σand p73αin the p53 gene mutation breast cancer45 of 76 breast cancers were p53 positively expressed (59.2%). In the 45 breast cancer patients, the rate of both p73αand 14-3-3σpositively expressed was 61.1% (11/18), and the rate of both p73αand 14-3-3σnegatively expressed was 78.57% (22/28), positive correlativity was found(r=0.384,χ2=6.490, p=0.013).6 Relationship between the protein expression of p53 and p73αin the breast cancer tissuesPositive rate of p73αwas 42.2% (19/45) in the p53 positive group, and the rate was only 19.35% (6/31) in the p53 negative group, and they were positive correlation (r=0.239,χ2=4.919, p=0.027).Conclusions: 1 p73αprotein was more hyper-expressed in the normal breast tissues and tissues besides tumor than in the tumor tissues, and negative correlation was found between the hyper-expression of p73αprotein and the haemal tube tumor embolus of the tumor tissues, especially in the patients whose ER and PR were all positive. It indicated that as an anti-oncogene, the attenuation of p73αexpression had something to do with the formation and development of breast tumor.2 The negative regulation factor of cell cycle 14-3-3σhad an attenuation tendency in the course from normal breast tissues to hyperplasia and to cancerization, and it was negative correlation with the histology grades and HER-2, Which indicated that it maybe an important suppressor factor in the carcinogenesis and development of breast cancer.3 Hyper-expression of p53 protein and the expression of p73αgene were positive correlation, and positive correlation was also found between the expression of p73αand 14-3-3σgene in the p53 gene mutated breast cancer tissues, which indicated that when p53 gene mutated, 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.PartⅡP73αup-regulated 14-3-3σin adriamycin-treated, p53-mutant human breast cancer cell lineObjective: To investigate the regulative function of p73αto the regulator of cell cycle 14-3-3σafter DNA injury induced by ADR in the p53 mutant human breast cancer cell line MDA-MB-436, and confirm the correlation between p73αand 14-3-3σ.Methods: MTT assay, Transfection, RT-PCR, Western-blot, immuno- precipitation were used to analyze the gene and protein expression of p53, p73αand 14-3-3σbefore and after gene transfection.Results:The survival rate of MCF-7 cell were respectively 50.20% and 16.58% 24 or 48 hours after ADR-treated, and the rate of MDA-MB-436 were 99.20% and 98.62% (p< 0.01), that is, p53 mutated cell line was drug fast to ADR; In the p53 mutated cell line, the expression of p73αgene up-regulated slightly, while the expression of 14-3-3σwas down-regulated significantly after DNA injury induced by ADR; the expression of 14-3-3σsifnificantly up-regulated after exogenous p73αwas transmitted into MDA-MB-436 cells, and the survival rate of MDA-MB-436 cell were respectively 58.20% and 24.66% 24 or 48 hours after ADR-treated (p<0.01).Conclusions: P73αfunctionally replace p53 in triggering its downstream target 14-3-3σwith higher expression, by which tumor growth will be controlled in p53-mutant human breast cancer cell line, then provided a new way to the treatment of p53 dependent breast cancer.PartⅢEffect of 14-3-3σon the Transcriptional Activity of p73αObjective: p53 is the main regulator of 14-3-3σ. The expression of 14-3-3σis induced by p53,while 14-3-3σstabilizes p53 and enhances its tanscriptional activity. P63 and p73, which are the members of p53 family have some functions similarly to p53. This study was designed to investigate the effect of 14-3-3σon the transcriptional activity of p73α. Methods:Luciferase reporter assay,RT-PCR,Western-blot and colony formation assay were used to study the effect of 14-3-3σon the transcriptional activity of p73αgene in p53-deficient human lung carcinoma H1299 cells and p53-mutant human breast cancer MDA-MB-436 cells.Results:Luciferase reporter assay showed that among the control group, the group transfected with p73αas well as the group transfected with p73αand 14-3-3σ, the luciferase activities induced by bax and p21WAF1 promotors were significantly different in H1299 cells (p<0.01). And it was shown a dose-dependent manner between luciferase activities and 14-3-3σtransfection (p<0.01). RT-PCR and Western-blot also showed that among the control group, the group transfected with p73αas well as the group transfected with p73αand 14-3-3σ, the expressions of bax and p21WAF1 were significantly different (p<0.01). Colony formation assay showed that in MDA-MB-436 cells, the cells died differently among the three groups after screened by G418 (p<0.01).Conclusions: 14-3-3σcan enhance the transcriptional activity of p73 in dose- dependent manner.Part IV The effect of 14-3-3σon p73αstability in p53-mutant breast cancer cell lineObjective: To investigate the effect of 14-3-3σon p73αstability in p53-mutant breast cancer MDA-MB-231 cell line.Methods:In this research, transfection, RT-PCR, Western-blot, protein decay analysis by cycloheximide were used to study the effect of 14-3-3σon p73αstability.Results:RT-PCR showed that the ratio of p73 and GAPDH is 0.635 after transfected with 1μg p73α; the ratios of p73αand GAPDH are 0.643 and 0.631 after transfected with 1μg p73α+1μg 14-3-3σas well as 1μg p73α+2μg 14-3-3σ, and no significant difference was found between each ratios (p>0.05).Western blot show ed that the ratio of p73αand actin is 0.333 after transfected with 1μg p73α; the ratios of p73αand actin are 0.797 and 0.826 after transfected with 1μg p73α+1μg 14-3-3σas well as 1μg p73α+2μg 14-3-3σ, and significant differences were found between each ratios (p<0.01). After transfected with p73αand cycloheximide treatment for 0h, 1h, 2h, 4h, 6h, the ratios of p73αand actin are 0.075, 0.166, 0.124, 0.100, 0.092; While after transfected with p73αand 14-3-3σand then cycloheximide treatment for 0h, 1h, 2h, 4h, 6h, the ratios of p73αand actin are 0.963, 0.244, 0.244, 0.234, 0.185, and significant differences was found (p<0.01).Conclusions: 14-3-3σhas no effect on p73αstability on transcriptional level; while 14-3-3σcan increase the stability of p73αon protein level.Part V Internal Empirical study of the restrain function of p73αand 14-3-3σto the growth of breast tumorObjective:To investigate the restrain function of p73αand 14-3-3σto the growth of breast tumor in vivo, and further to confirm the function mechanism of 14-3-3σ, and the regulative function of p53 and p73αgene to 14-3-3σ.Methods:Transfected exogenous gene of cells and transplanted model of breast cancer of immunologic deficiency mice were used. According to the different experimental groups, we imported p53,p73αand 14-3-3σinto the p53-mutate cell line MDA-MB-231. We observed the growth of tumors after different MDA-MB-231 groups were transferred into feamale athymic mouse and the interference of therapeutic dose of ADM.Results: Tumors which were confirmed as breast cancer by pathology, grew in the 14-3-3σtransfected 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 14-3-3σhave both activity and inactivity states. The activity state can inhibit the growth of tumor, but 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.