Study Of The Relationship Between The Gene Silence Of Breast Cancer Susceptibility Gene, BRCA1 And The Pathogeny Of The Sporadic Breast Cancer

Study of the Relationship between the Gene Silence of Breast Cancer Susceptibility Gene, BRCA1 and the Pathogeny of the Sporadic Breast CancerObjectiveBreast cancer is a malignant tumor threaten the healthy of female, it can be divided into genetic and sporadic two kinds of breast cancer. Most of them (95%) were sporadic breast cancer. The BRCA1 (the breast cancer susceptibility gene 1) was localized by linkage analysis of multiple families affected by early-onset breast and ovarian cancer. It was cloned in 1990s, mutation in BRCA1 are responsible for nearly 80% of familial breast cancer, The research have found that decreased mRNA levels and the low expression of BRCA1 protein in sporadic breast cancer, while the reason haven't clarified. By the progress of research on the epigenetic, there are hypothesis consider the hypermethylation of BRCA1 promoter leads to the low expression of BRCA1 protein. So in our research, we study the methylation statues of 5' CpG island locating in the promoter region of BRCA1 and the correlation with the expression of BRCA1 protein; detect the copy number of BRCA1 in large series of sporadic breast cancer, discuss the relationship between the hypermethylation of BRCA1 promoter and the copy number of BRCA1, clarify the mechanism of BRCA1 gene silence and the role of BRCA1 gene in the carcinogenesis and progression of sporadic breast carcinoma.MethodsWe examined the expression of BRCA1 protein gene in tissues embedded by paraffin of 98 Chinese sporadic breast cancer, as well as 12 benign breast disease tissues by using immunohistochemistry (IHC); analysis the relationship between the expression BRCA1 and the clinic parameters (such as the patient's age, type of histology and histological grade, status of Axillary's lymph nodal, the expression of estrogen receptor and progesterone receptor); and the expression of other regulation genes, such as the oncogenes (C-myc, bcl-2 and HER-2) and the tumor depression gene (p53).Assay the methylation status of promoter region of BRCA1 gene in frozen tissue of 98 sporadic breast cancer tissues, as well as 12 benign breast disease tissues by methylation specific PCR (MSP), Analysis the correlation between hypermethylation of the BRCA1 promoter and the decreased expression of BRCA1 protein in sporadic breast cancer. We detected the copy number of BRCA1 and CEP17 by interphase FISH (fluorescence in situ hybridization); calculated the ratio of copy numbers of BRCA1/CEP17, in the tissues of benign breast disease and those tissues with hypermethylation of BRCA1 promoter, or without. We analyzed the relationships between hypermethylation of the BRCA1 promoter and the delete of BRCA1 gene and CEP17.Results1. The expression of BRCA1 protein in the breast tissues: BRCA1 protein was localized in the nuclei of ductal and lobular epithelia in 12 benign breast disease samples. In all 98 sporadic breast cancer samples, 52(53.1%) samples have BRCA1 protein expression exclusively in nuclear and 46(46.9%) samples no nuclei expression of BRCA1 protein.2. The Relationship between The expressions of BRCA1 protein and clinic pathology parameters: There are 37.0% (10/27) samples have BRCA1 protein expression in the category of histological stageⅢ, while which are 72.4% (21/29), 50.0% (21/42) in the stageⅠandⅡ; the ratios of positive expression of BRCA1 protein in the nuclei were 55.2% (16/29), 40.5% (17/42) and 11.1% (3/27) according to the histological stages (Ⅰ～Ⅲ). Loss expression of BRCA1 protein had significant correlation with high histological stages (Ⅰ～Ⅲ)(P＜0.01). There are 34.8% (17/45) tissues have BRCA1 protein positive expression in the group that companying the axillary's lymph nodal metastasis, lower than that of 66.0% (35/53) in the group haven't the axillary's lymph nodal metastasis. Loss expression of BRCA1 protein had significant correlation with axillary's lymph nodal metastasis (P＜0.01). The loss expression of BRCA1 protein had no significant correlation with the histological types and the expression protein of ER, PR proteins.3. The correlation between the expression of BRCA1 protein and related regulation proteins: The ratio of positive expression of BRCA1 in the nuclei was 45.2% (14/31) in the breast cancer tissues low express the HER-2 protein. Which were 11.1% (1/9) and 0.0% (0/5) in the breast cancer tissues middle and high express the HER-2 protein. No expression of BRCA1 protein companied with the more expression of HER-2 protein (P＜0.05). The low expression of BRCA1 had no significant correlation with the expression of bcl-2, C-myc, p53 proteins.4. The detection of the hypermethylation of BRCA1 promoter in breast tissues: No methylation of BRCA1 promoter were found in all 12 benign breast disease samples, 15 cases in 98 sporadic breast cancer samples (15.3%) were detected hypermethylation of BRCA1 promoter by Methylation Sensitive PCR.5. The relationship between promoter hypermethylation and the expression of BRCA1 protein and related regulation proteins: 15 cases in 98 sporadic breast cancer samples were detected hypermethylation of BRCA1 gene in promoter region, all of them loss expression of BRCA1 protein. There are 46 cases loss expression of BRCA1 protein, so the hypermethylation of BRCA1 promoter induced loss expression occupy the 32.6% (15/46). There are no directed correlation between the hypermethylation of BRCA1 protein and the expression of related regulation proteins (bcl-2, p53, HER-2 and c-myc).6. The relationship between promoter hypermethylation of BRCA1 and the clinic pathologic parameters: There were 29.6% tissues had hypermethylation of BRCA1 promoter in the histological stageⅢ, while which were 3.4% in the stageⅠ, the difference was significance (P＜0.01). There were 24.4% (11/45) tissues have hypermethylation of BRCA1 promoter in the group that companying the axillary's lymph nodal metastasis, higher than that 7.5% (4/53) in the group haven't the axillary's lymph nodal metastasis. The hypermethylation of BRCA1 promoter had significant correlation with axillary's lymph nodal metastasis (P＜0.01).7. The correlation between the copy number of BRCA1 gene and the hypermethylation of BRCA1 promoter: In the 3 cases of benign breast disease, 12 cases of breast cancers with hypermethylation of BRCA1 promoter and 5 cases without the hypermethylation of BRCA1 promoter, the mean copy number of BRCA1 in methylated cases (1.41±0.18) was lower than that (1.98±0.09) of in unmethylated cases (P＜0.05) by interphase FISH. The mean one copy of BRCA1 was (0.84±0.03)% in the benign breast disease, The proportion of one copy of BRCA1 was (22.04±1.06)% in breast cancers with the hypermethylation of BRCA1 promoter, higher than that of (2.56±0.32)% in the breast cancers without the hypermethylation of BRCA1 promoter, the difference was significant (P＜0.01). The proportion of gain copies of BRCA1 (20.86±1.92)% in the breast cancers without the hypermethylation of BRCA1 promoter, higher than that (1.47±0.20)% of in the breast cancers with the hypermethylation of BRCA1 promoter(P＜0.01).8. The correlation between the ratio of BRCA1/CEP17 and the hypermethylation of BRCA1 promoter: The mean copy number of CEP17 was 2.33±0.17 in the benign breast cancer, which was 1.92±0.19 in the methylated cases lower than that of (2.39±0.46) in the unmethylated eases, while the difference haven't significant. The ratio of BRCA1/CEP17 in the methylated cases (0.74±0.11) was slightly lower than in the unmethylated eases (0.85±0.13), and compared to the mean (0.88±0.05) in the benign breast cancer. There wasn't difference significant.Conclusions1. All of the expression of BRCA1 protein was located to nuclei in benign breast disease samples; the nuclei expression of BRCA1 protein was reduced in the sporadic breast cancer, related to higher histological grade, the axillary's lymph nodal metastasis. Low nuclei expression of BRCA1 protein is also correla- ted to amplification of HER-2 protein.2. Hypermethylafion of BRCA1 promoter leads to low expression of the BRCA1 protein in sporadic breast cancer, the mechanism might be hypermethylation of BRCA1 promoter leads to the loss copy of BRCA1 and CEP17.