| Background:Malignant tumor has become one of the most serious diseases which threatened the health of people.Seriously,the incidence and mortality of overall cancer are rising as soon as the situations become more serious among the contamination,the pressure and the rhythm,which has been the biggest cause of death in patients.According to the latest report of World Health Organization,there are thousands of women came to death because of breast cancer,which is the most common disease than the other malignant tumor.Although the incidence of breast cancer in our country is low,the 2012 China cancer registration update released that more and more women under the threat of breast cancer along with the higher incidence and mortality trends with cancer which were happened in the past 20 years.Therefore,it is necessary to study on the occurrence and development of breast cancer as well as to the research into the mode of the multi-line treatment of breast cancer.Studies showed that,carcinogenesis and development of malignant tumor had multiple steps and was induced by multiple genes,which were studied from genetics and epigenetic.As one of the tumor suppressor p53 protein family,p73 maps to the chromosome 1p36 region which has two distinct promoters coding for two major isoforms,the pro-apoptotic TAp73 isoforms and the anti-apoptotic ?Np73 isoforms,respectively.The extrinsic promoter(PI)located upstream to exon 1,which gives rise to the transactivation domain(TA)-containing(TAp73)isoforms(?,?,?,?,?,?,?,?,?1)and the intrinsic promoter(P2)located in intron 3,which controls the transcription of TA-deprived(?Np73)isoforms.Numerous studies indicate that the P1 promoter generates the TAp73 isoform containing transactivation domain(TA)could activate the transcription of numerous p53 target genes and inhibit cell growth in a p53-like manner,such as cell cycle arrest and apoptosis.Functional report shows that mice with a selective deficiency of TAp73 develop spontaneous tumors and are more sensitive to chemical carcinogenesis.Encoded by P2 promoter,the ?Np73 isoform,which is lacking the TA domain,enable to inhibit the oncogenic activity of p53 and TAp73 by competing DNA-binding sites or oligomerizing with the full length proteins.Overexpression of ?Np73 in many human cancers has been shown to inhibit apoptosis,and increased levels of ?Np73 in primary tumors have been shown to correlate with poor prognosis.TAp73 activation mediated up-regulation of ?Np73 in cancer cells,while up-regulation of ?Np73 inhibited TAp73 expression,suggesting a feedback network between TAp73 and ANp73.When the balance between TAp73 and?Np73 was broken and ANp73 was up-regulated,the body would undergo malignant lesions.The relative expression of these two proteins is involved to the prognosis of several cancers.Therefore,the balance between TAp73 and ?Np73 finely regulates cellular sensitivity to death.DNA methylation is an epigenetic regulatory mechanism,establishing long-term gene silencing during development and cell commitment,proceeding extensively at CpG-rich regions that,in many instances,are located at promoter regions.Unlike p53,the p73 is rarely mutated in many human cancers,whose loss expressions are related with DNA methylation at CpG-rich regions.Corn PG et.al indicated there was no evidence for p73 exon 1 methylation in normal tissues,while p73 was aberrantly methylated in approximately 30%of primary acute lymphoblastic leukemias(ALLs)and Burkitt's lymphomas as well as methylation was associated with transcriptional loss of p73 in both leukemia cell lines and primary ALLs.The results showed that methylation of p73 is a frequent event in specific types of hematological malignancies and suggested that epigenetic silencing of p73 could have important consequences for cell-cycle regulation.However,Daskalos A et.al demonstrated that while P1 hypermethylation-associated reduction of TAp73 mRNA levels is relatively infrequent,the P2 hypomethylation-associated over-expression of ANp73 mRNA is a frequent event,particularly among squamous cell carcinomas.Therefore,different promoter region methylation of p73 gene eventually caused the different biological effects in malignant tumor.Formerly researches about DNA methylation are mainly concentrated in P1 promoter,relatively little in P2,and then Tang Lin et.al found abundant CpG islands in P1 and P2 were predicted by Online softwares which provide a scientific reference for further study,and 5-aza-dC(5-aza-2'-deoxycytidine)as a DNA Methyltransferase(DNMTs)inhibitor was used to epigenetic drug.Recent report has showed that methylation of p73 gene is re-expression as a consequence of promoter demethylation by 5-aza-dC in hematological malignancies such as lymphoma,acute lymphoblastic leukemia(ALL)as well as in some solid tumors such as lung cancer,gastric carcinoma and cervical cancer.The transcription factors binding sites in the promoter regions of p73,lead to functional differences between transcription products.For instance,Yu J et.al found P1 promoter has five distinct Egrl-binding sites,each contributing to the transcriptional upregulation of TAp73 by Egrl in several cell types.While P2 promoter transcribes ?Np73,is not induced by Egrl.Logotheti S et.al indicated Spl binds to the external promoter of the p73 gene and induces the expression of TAp73y in lung cancer.We found the transcription factors binding sites AP-1 in P2,and Vikhanskaya F et.al demonstrated a novel and unexpected role of p73 in augmenting AP-1 transcriptional activity through which it supports cellular growth.At the same time,using bioinformatic analysis to predict transcription factors binding sites in p73 promoters,we found that both P1 and P2 promoter have the putative binding sites for nuclear factor erythroid 2-related factor 2(Nrf-2),which is a member of CNC(cap 'n'collar)and plays an important role in determining the fate of the cell,impacting fundamental biological processes such as proliferation,apoptosis,angiogenesis and metastasis.It was also been reported that the expression of Nrf-2 was suppressed by methylation of certain CpG sites in the Nrf-2 gene promoter.Meanwhile,the re-expression of Nrf-2 occurred as a consequence of promoter demethylation by 5-Aza-CdR.Objective:In this study,we investigate 5-aza-dC treatment effect of the breast cancer cells growth along with cell cycle arrest and apoptosis,and reveal the promoter CpG methylation state in human breast cancer and the transcriptional regulation mechanism of p73 promoters by Nrf-2 in cell lines,mice model and tissue.Provide the basis understanding of 5-aza-dC treatment that might serve as predictive biomarkers of response or functional targets for therapeutic intervention.Methods:1.To observe 5-aza-dC effect of the growth of breast cancer cell lines,cell cycle arrest and apoptosis by MTT assay and Flow cytometric analysis.2.To observe 5-aza-dC effect of the DNMTs in breast cancer cell lines by DNMTs activity analysis.3.To detecte methylation state of CPG islands in P1 and P2,and observe demethylation by 5-aza-dC in MCF-7 cell line by pyrosequencing assay.4.To observe the expression of TAp73 and ?Np73 induced by 5-aza-dC in MCF-7 cell line by reverse transcription PCR(RT-PCR),Western blot analysis and immunofluorescent staining assay,.5.To predict transcription factors binding sites in p73 promoters and understand the possible mechanisms of Nrf-2 regulates TAp73 and ?Np73 expression with 5-aza-dC treatment in MCF-7 cell line by Bioinformatic analysis,CHIP assay and Quantitative Real-Time Polymerase Chain Reaction(qRT-PCR).6.To observe the TAp73 and ?Np73 expression induced by Nrf-2 in MCF-7 cells line by transiently transfected assay and Western blot analysis,.7.To observe the TAp73 and ANp73 expression induced by Nrf-2 with 5-aza-dC treatment in Nrf-2 gene knock-out mice model by Western blot analysis.8.To further explore the particular relationship of TAp73,?Np73 and Nrf-2 expression in breast cancer tissue microarrays by immunohistochemistry staining assay.Result:1.5-aza-dC induces cell proliferation inhibition along with cell cycle arrest and apoptosis in breast cancer cell lines.(P<0.05)2.5-aza-dC inhibites DNMTs activity in breast cancer cell lines.(P<0.05)3.5-aza-dC cause demethylation in P1 and P2 which are detected enrich CPG islands in MCF-7 cell line.4.5-aza-dC induces the expression of TAp73 and inhibits the expression of ?Np73 in MCF-7 cell line.(P<0.05)5.Nrf-2 can specifically bind to the TAp73 and ?Np7 at the one binding sites in P1 promoter and three binding sites in P2 promoter,and the Nrf-2 binding to TAp73 was enhanced,while the Nrf-2 binding to ?Np73 was weakened in response to treatment with 5-aza-dC.6.Nrf-2 transfection induced TAp73 and ?Np73 expression in MCF-7 cell line.7.The TAp73 and ANp73 protein levels were significantly decreased in mammary gland of Nrf-2-/-mice model when compared with Nrf-2 +/+ mice model.However,Nrf-2 induced ANp73 expression was abolished with 5-aza-dC treatment.8.A decreased expression of TAp73 and an increased expression of ANp73 and Nrf-2 in breast cancer tissue than the corresponding NATs,and significant negative correlation were found between TAp73 and ANp73 expression in this breast cancer tissue-array.Conclusion:5-aza-dC induced cell growth inhibition,G1 cell cycle arrest and apoptosis in breast cancer cells were dependent on an up-regulated TAp73 expression and a down-regulated ANp73 expression.We also defined that Nrf-2 and promoter methylation cooperatively governs the transcriptional regulation of p73 promoters.On one hand,5-aza-dC induced TAp73 and ?Np73 expression by DNA methylation in P1 and P2.On the other hand,5-aza-dC induced an up-regulated TAp73 expression and a down-regulated ANp73 expression,which correlated with an increasing Nrf-2 binding to P1 while a decreasing Nrf-2 binding to P2.Moreover,our findings about a decreased TAp73 and an increased ?Np73 and Nrf-2 expression and the negative correlation between TAp73 and ?Np73 expression in breast cancer tissue maybe one of the important factors in the development of breast cancer. |
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