| Breast cancer is one of the most common malignant tumor in women's health,its incidence is gradually increasing trend.In China in recent years,the incidence of breast cancer in women malignant tumor,and disease appear younger.With the deepening of the biological behavior of breast cancer,and the transformation of the concept of treatment and update,to understand the pathogenesis of breast cancer,early diagnosis,comprehensive treatment for breast cancer and after the play an important role.DNA replication is a critical step in normal cell division and proliferation,DNA polymerase ? is an important role in cellular DNA replication enzymes,which polymerase and exonuclease activity are located on the catalytic subunit p125.The occurrence an development of breast cancer is composed of multiple factors involved in cell cycle regulation of pathological changes gradual process,P53 as an important tumor suppressor gene is closely related to the occurrence of breast cancer.By bioinformatics analysis,P125 gene encoding the POLD1 promoter is rich in CpG Island,and containing transcription factors p53 binding sites,these sites coincide with a number of CG site.Abnormal DNA methylation is associated with malignant cell proliferation,Peculiarity of transcription factor binding sites in the cell has the CpG loci,the existing research is pointed out that the abnormal of these loci promoter methylation is associated with the pathogenesis of breast cancer and other tumors.To understand the methylation level of POLD1 promoter and the abnormal regulation of DNA replication,it can provide a new idea for the prevention and treatment of breast cancer.At present,there is no research to explain how the expression of P125 in breast cancer is regulated by cell cycle.This study proposed by collecting clinical breast cancer tissue samples and establishing the model of breast cancer cells,Using genomics,proteomics and molecular biology technology to explore the regulatory factor p53 and related transcription factors,influence the expression of P125 in cell cycle regulation,transcriptional regulation and epigenetic regulation on,Further to explore the mechanism of P125 abnormal expression in breast cancer,and explore the P125 on the occurrence,development and prognosis of breast cancer and its effects on cellular malignant phenotype.Part ? P125 expression in breast cancer tissue and the correlation of promoter methylation and P53 expressionObjective:Discussion P125 promoter methylation and expression levels in breast cancer of P125 and P53Methods:Collect from the Affiliated Tumor Hospital of Guangxi Medical University from October 2012 to December 2013 the pathological diagnosis of breast cancer and adjacent tissues in 100 cases(refers to from the tumor tissue adjacent to carcinoma is larger than 2cm the tumor tissue).Pyrosequencing detection of breast cancer and adjacent tissue methylation level of p125 gene encoding POLD1 promoter.The qRT-PCR and Western blot detection POLD1 and P125 protein expression in breast carcinoma and adjacent tissues.Using qRT-PCR and Western blot to detect the expression of P53 in breast carcinoma and adjacent tissues.Results:The methylation rate of POLD1 gene promoter in breast cancer tissues was(52.51+1.86)%,significantly higher than that in adjacent tissues(33.47+1.59),and the difference was statistically significant(P<0.05).Fluorescence quantitative PCR showed POLD1 gene expression in breast cancer was significantly higher than that in the adjacent tissues,Western blot showed that in breast cancer P125 protein expression was significantly higher than the adjacent group,which is consistent with the results shown in PCR results,the differences were statistically significant(P<0.05).P53mRNA in breast cancer tissue and tissue adjacent to carcinoma rt-pcr result is shown in the expression of breast cancer tissue below the tissue adjacent to carcinoma,Western blot P53 protein expression and the experimental results indicate the PCR results is consistent,the difference was statistically significant(P<0.05).Conclusion:P125 in breast cancer tissue and high expression,regulation factor P53 can combine with POLD1 promoter site,may affect POLD1,promoter methylation leels were associated with the pathogenesis of breast cancer.Part ? On breast cancer cells POLD1 promoter methylation of lentivirusmediated overexpression of P53Objective:Construction of P53 gene overexpression lentivirus vectors and infection of human breast cancer cell line MCF-7,and its influencing on breast cancer cells of p125 gene encoding POLD1 promoter methylation levels.Methods:Cultured human breast cancer cell line MCF-7 and normal human breast epithelial cells MCF-10A,Using qRT-PCR and Western blot detection of breast cancer cell line MCF-7 and normal breast epithelial cells MCF-10A expression levels of p53.The methylation level was detected in MCF-7 cells and MCF-10A cells of POLD1 promoter.Logarithmic growth phase in human breast cancer MCF-7 cells were inoculated into the culture plates,were infected with P53 gene overexpression lentivirus vector is transfected(rLV-P53);Infected with lentivirus vector as a negative control group(rLV-NC);Blank control group without any treatment.Sequencing pyrophosphate detection of P53 expression of MCF-7 turn stable cell lines POLD1 promoter methylation changesResults:Real-time PCR results showed P53 expression in human breast cancer MCF-7 cells is lower than normal human breast epithelial cells MCF-10A,Western blot results showed in MCF-7 P53 protein expression level was significantly lower than MCF-10A,which is consistent with the results shown in PCR analysis,differences were statistically significant(P<0.05).Pyrosequencing display of human breast cancer MCF-7 cell lines POLD1 promoter methylation was(57.9± 8.6)%,significantly higher than the normal human breast epithelial cells MCF-10A(15.6±6.2)%,the difference was statistically significance(P<0.05).Construction of lentivirus-mediated overexpression of P53 in breast cancer MCF-7 cells,the Pyrosequencing detection POLD1 promoter methylation rate,results showed overexpression of P53 MCF-7 cells(37.6 ± 5.6)%,significantly lower than human breast cancer cell line MCF-7(57.9 ± 8.6)%,the difference was statistically significant(P<0.05).Conclusion:P53 is closely related to the occurrence of breast cancer,and the POLD1 promoter methylation of p53 was significantly lower in the over expression of MCF-7 in breast cancer cell line.POLD1 promoter methylation level was down regulated,and may regulate the expression of P125 related.Part ? Effects of P53 overexpression on the biological characteristics of breast cancer cellsObjective:Construction of P53 gene overexpression lentivirus vectors and infection of human breast cancer cell line MCF-7,Explore the biological characteristics of breast cancer cells and molecular mechanisms of influence.Methods:Selected the constructed lentivirus infection P53 gene overexpression vector MCF-7 cells(MCF-7/P53)and infected with lentivirus vector MCF-7 cells(MCF-7/NC),cell proliferation assay using CCK8 observed proliferation.After Annexin V-PE/7-AAD staining after treatment detected changes in cell cycle and apoptosis by flow cytometry.Transwell migration and invasion assay to observe the migration and invasion.Results:P53 overexpression of stably transfected MCF-7 cell line MCF-7 and the empty virus-infected cell lines treated by CCK8 proliferation was detected,the display overexpression of MCF-7 cell proliferation was significantly lower than the control group P53 empty virus-infected MCF-7 cell.Analyzed by flow cytometry cell cycle,comparative analysis of G1,G2,S and value no significant difference.Analyzed by flow cytometry of apoptosis in each group,the display MCF-7/P53 early apoptosis rate was(10.8±1.1)%,compared with the control group MCF-7/NC early apoptosis rate(5.1±0.5)%,the difference was statistically significant(P<0.05),In the apoptosis of MCF-7/P53 is(11.6±1.3)%,while the control group MCF-7/NC was(5.3±0.6)%,the difference was statistically significant(P<0.05);but late apoptosis rate difference was not significant.Transwell cell migration assay showed that overexpression of P53 of MCF-7 cell line stably transfected migration was significantly lower than the control group.Transwell cell invasion assay results showed that overexpression of P53 of MCF-7 cell line stably transfected invasion ability than the control group MCF-7 cell lines infected air decreased,and the difference was statistically significant(P<0.05).Conclusion:Overexpression of P53 can inhibit the proliferation of breast cancer cells,reducing metastasis and invasion capacity of the cells can be induced early breast cell apoptosis.Part ? Regulation of p53 and Sp1,DNMT1 in breast cancer cells and its effect on p125Objective:To explore P53 and Spl,DNMT 1 regulatory mechanisms and its influence in the MCF-7 breast cancer cells P125 expression and promoter methylation levels.Methods:Using Western blot to detect MCF-7 human breast cancer cells and overexpression of MCF-7 in breast cancer cells P53(MCF-7/p53)expression level of Sp1 and DNMT1.Using Coimmunoprecipitation assay detecting the binding of P53 and Sp1.Dual Luciferase reporter gene assays and Electrophoretic mobility shift assays to detect SP1 and DNMT1 promoter binding conditions.Western Blot method was used to detect the expression level of P125 protein in human breast cancer cell MCF-7 and over expression of P53 in breast cancer cell line MCF-7.Results:Western blot analysis results showed Sp1 and DNMT1 proteins were expressed in MCF-7 and MCF-7/P53,and the blank MCF-7 contrast overexpression of of P53 MCF-7 cell line,protein expression levels of Sp1 and DNMT1 significantly reduced.Co-immunoprecipitation experiments results showed P53 protein in breast cancer cells can be combined with Sp1 protein.Dual luciferase reporter gene assays showed that the mutant DNMT1 gene promoter luciferase reporter gene activity compared to normal promoter luciferase reporter gene activity was inhibited significantly(P<0.05),By EMSA analysis of experimental results shows that Sp1 in breast cancer cells can be combined into DNMT1 promoter.Detection of P125 Western blot expression level,results showed that compared with MCF-7 over expression of P53 MCF-7 stable strain,the expression of P125 protein decreased significantly,indicating the expression level of P125 was regulated by P53.Conclusion:P53 protein in breast cancer cells can be combined with Spl proteins,and regulate the expression of Sp1,Sp1 can be used as transcription factor binding to the DNMT1 promoter specifically,affect p125 promoter methylation level,and then regulate the expression of p125. |
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