Study Of The Relationship Between Promoter Methylation Of BRCA1 And The Sporadic Breast Cancer

Objective: Breast cancer is one of malignant tumors that threaten the health of women.Breast cancer can be divided into familial breast cancer and sporadic breast cancer,most of them(about 95%)were the sporadic breast cancer,familial breast cancer accounted for 5%.The mutation of BRCA1 gene is important for familial breast cancer.However,BRCA1 gene sequence was complete in sporadic breast cancer,mRNA of the gene decreased obviously.This indicates that BRCA1 gene with other mechanisms involved in sporadic breast cancer.The present studies suggest that tumor suppressor gene DNA methylation plays an important role in the process of normal cells into tumor cells and the enhanced invasion of tumor cells.Studies have shown that BRCA1 gene promoter methylation is closely related with the loss of expression of BRCA1 and sporadic breast cancer initiation and progression.BRCA1 promoter methylation in Sporadic triple negative breast cancer was higher than other types.The purpose of this experiment is to detect BRCA1 promoter CpG island methylation level,to compare methylation difference of different types in sporadic breast cancer,to explore the relationship between BRCA1 promoter methylation and clinical characteristics of sporadic breast cancer and provide guidance for the prognosis.Methods:1 Sample collectionThe collected samples are 79 cases of breast cancer tissues that received treatment at the Breast Center of Fourth Hospital of Hebei Medical University from April 2014 to March 2015.All samples whose age were from 27 to 81 years old are females without a family history of breast cancer and other tumor history.The median age was 53 years.All patients were confirmed to be breast cancer by biopsy before operation,without radiotherapy,chemotherapy and endocrine therapy.According to Breast cancer Estrogen receptor(ER)and progesterone receptor(PR)and Her2 protein immunohistochemical detection standard by ASCO and CAP in June 2013,2013 St.Gallen expert consensus and the results of immunohistochemistry,the 79 patients were divided into four subtypes:(1)22 cases of patients with Lumimal A breast cancer,which were the high expression of ER/PR positive and PR(?20%),Her2 negative and low expression of Ki-67(<14%);(2)19 cases of patients with Lumimal B breast cancer,which were ER/PR positive.If Her2 was negative,Ki-67 was high expression or PR was low expression;if Her2 was positive,Ki-67 could be any states.(3)18 cases of patients with Her2 Positive breast cancer,which were Her2 positive,ER and PR negative.(4)20 cases of patients with triple negative breast cancer,which were ER,PR and Her2 negative.Record clinical and pathological datum in detail.2 Experimental ProcessThe genomic DNA was extracted using TIANamp Genomic DNA Kit.DNA was modified by bisulfite.All unmethylated cytosines were converted to uracils,while methylated cytosines unchanged.Primer pairs amplifying a 195 base pair(bp)product of BRCA1 promoter CpG Island were designed as follows: forward primer 5'-TATTTTTGTGGGGTGAATTTAATATG-3' and reverse primer 5'-CCCTCAACCCCAATATTTATTATTT-3'.Uracil all converted to thymine by PCR amplification.The PCR product was cloned into T vector.Then The plasmid DNA extracted was sent to sequencing company.3 Statistical AnalysisThe data was analyzed by SPSS19.0 statistical software,p<0.05 was considered statistically significant.Measurement data was described by the mean�standard error of the mean((?)�s).Count data was described by frequency(n%).Count data were analyzed by Chi square test.Comparisons between the two groups were compared by Kruskal-Wallis test,and the U Mann-Whitney U test was used for the comparison between groups.Results:1 Comparison of clinical biological characteristics of different molecular subtypes in breast cancerAge,T stage(tumor size),N stage(the number of lymph node metastasis),TNM stage,pathological type and histological grade in the patients of each group were compared by the chi square test.The results were as follow:There was a significant difference(X~2=10.462,P=0.015<0.05;X~2=10.181,P=0.017<0.05;X~2=7.110,P=0.029<0.05)in pathological types between triple negative breast cancer and other subtypes(Luminal A,Luminal B and Her2 positive).The pathological types in riple negative breast cancer were mainly invasive ductal carcinoma and medullary breast carcinoma.The pathological type in other subtypes was mainly invasive ductal carcinoma.There was a significant difference(X~2=7.819,P=0.020<0.05;X~2=24.130,P=0.000<0.05;X~2=15.724,P =0.000<0.05)in histological grade between Luminal A and other subtypes(TNBC,Luminal B and Her2 positive).The histological grade in Luminal A was mainly II grade.The histological grades in other subtypes were mainly III grade.There was a significant difference(X~2=7.441,P=0.006<0.05)in histological grade between Luminal B and Her2 positive.The histological grade in Luminal B was mainly II grade and III grade.The histological grades in Her2 positive were mainly III grade.There was a significant difference(X~2=10.462,P=0.015<0.05)in pathological types between Triple negative group and Luminal A group.The pathological types in Triple negative group were mainly invasive ductal carcinoma and medullary breast carcinoma.The pathological type in Luminal A group was mainly invasive ductal carcinoma.There was a significant difference(X~2=15.724,P=0.000<0.05)in histological grade between Triple negative group and Luminal A group.The histological grades in Triple negative group were mainly II grade and III grade.The histological grade in Luminal A group was mainly II grade.There was no significant difference in other statistical indicators(P >0.05).2 The description of methylation status of 12 CpG lociCpG site-842 and CpG site-782 had the highest methylation frequencies(65.8%).This showed methylated status of CpG site-842 and-782 was common.CpG site-742 had the lowest methylation frequencies(10.1%).This showed methylated status of CpG site-742 was rare.3 Comparison of the difference of methylation rate of 12 CpG loci in each subtypesMethylation rate of CpG site-881 in Luminal B group was higher than TNBC and Her2 positive(X~2=8.046,P=0.005<0.05;X~2=4.937,P=0.026<0.05).Methylation rates of CpG site-764 in TNBC were higher than Luminal A(X~2=8.066,P=0.005<0.05).Methylation rate of CpG site-742 in Triple negative group was higher than Luminal A and Luminal B(X~2=7.700,P=0.006<0.05;X~2=6.736,P=0.009<0.05).There was no significant difference in other CpG sites(P >0.05).4 Comparison of the methylation rate of four subtypesThere was no significant difference by comparison of the methylation rate of four groups(P=0.954>0.05).5 Comparison of the methylation rate between TNBC and other subtypes(Luminal A,Luminal B and Her2 positive)There was no significant difference by comparison of the methylation rate between TNBC and the other subtypes(P=0.642>0.05).6 Comparison of the relationship between sample methylation rate and clinical data(age,T stage,N stage,TNM stage,pathological type and histological grade)There was no significant difference by comparison of the relationship between sample methylation rate and clinical data(P >0.05).There is no correlation between methylation status and the clinical data.Conclusions:1 Methylation of BRCA1 promoter CpG loci-842 and loci-782 may be associated with sporadic breast cancer.Methylation rates of CpG site-764 and CpG site-742 in Triple negative group were higher than Luminal A group.Methylation rate of CpG site-742 in Triple negative group was higher than Luminal B group.Site-764 and site-742 methylation may be associated with sporadic triple negative breast cancer.2 Methylation rates of BRCA1 gene promoter(-908~-714)were not significantly different between sporadic triple negative breast cancer and other types of sporadic breast cancer(Luminal A,Luminal B,Her2 positive type).3 The methylation rate of BRCA1 promoter in sporadic breast cancer is irrelevant to age,tumor size,lymph node metastasis,TNM stage,pathological type and histological grade.