Promoter Region Methylation Of BRCA1 Gene In Sporadic Breast Cancers And Hyperplastic Breast Tissues

Background and objective: Breast cancer is one of the most frequently diagnosed severe female diseases. The incidence of breast cancer has been increasing since the industrilization of last century. It is crucial to diagnose and treat breast cancers in early stages. Breast hyperplasia, especially atypical hyperplasia is closely associated with breast cancer. There are morphologic similarities and epidemic correlations between breast hyperplasia and cancer, which suggests that they might have similar genetic or epigenetic alterations. According to modern molecular biological theory, a series of genetic or epigenetic alterations, especially the activation of oncogenes and the inhibition of tumor-suppressor genes have major contributions to the development of cancers. BRCA1, the first identified breast cancer susceptibility gene, is located on chromosome 17Q by linkage analysis of multiple families affected by early-onset breast and ovarian cancer in 1990. It was estimated that about 5%-10% of all breast cancer cases might be caused by inherited predisposition. Early genetic linkage analysis suggested that BRCA1 would be responsible for 45% the site-specific breast cancer family and the majority of breast-ovarian cancer family. Although mutations in BRCA1 are responsible for nearly 80% of familial breast cancer, it's involvement in the majority of breast cancers, which are sporadic and not familial, has not been demonstrated clearly. Decreased mRNA levels or aberrant subcelluar location of BRCA1 have been identified in sporadic breast cancer tissues. Aberrant methylation of the CpG island in the promoter region of BRCA1 gene has been considered as an important mechanism for the decline of gene expression. Our research aims to investigate normal and aberrant methylation of the CpG island in the promoter region of BRCA1 gene, to evaluate their significance in human sporadic breast cancer, clinical early diagnosis, and molecular therapy.Methods:A total of 23 fresh samples from patients with sporadic breast cancer underwet surgical treatment containing invasive carcinoma and para-tumor hyperplasia, 6 fresh samples of simple hyperplasia of breast.and 5 fresh samples of blood from healthy adult women were collected from 3 hospitals.Target genomic DNAs were extracted byusing the method of hydroxybenzene-chloroform. Target DNA was modified by sodium bisulfite, converting all unmethylated cytosines to uracil, and then subject to a novel sensitive method detecting DNA methylation. MSP(methylation specific PCR)combining with Nested-PCR was conducted with primers specific for methylated and unmethylated DNAs. The status of the CpG islands of BRCA1 gene in the promoter region was analyzed by the modified MSP assay. DNA sequencing were applied to evaluate the status of CpG hypermethylation in several specimens including cancer tissue, para-cancer tissue and simple hyperplasia tissue. The amplified segments of 3 specimens related to the primer M296-1 and M296-2 were cloned and then sequenced by using an automated fluorescence based cycle sequencer (ABI PRISM 3100) according to the manufacturer's instructions. The results and data of pathological characteristic were statistically analyzed using the software of SPSS10.0. The sequence was edited using the software of DNASTAR.Results: 1. In 23 sporadic breast cancer tissue, 65.22% showed aberrant methylation of the promoter region of 5'CpG island of BRCA1 gene. In 23 para-tumor tissues, 11 showed aberrant methylation of the 5'CpG island of BRCA1 gene. In the 15 cases of aberrant methylation in the promoter region of BRCA1 gene of breast cancer patient, 73.33% (11/15) of the 15 cases showed aberrant methylation in the promoter region of BRCA1 gene in the para-tumor hyperplasia tissues. 2.1n the 6 cases of simple hyperplasia tissues of breast, 2 cases showed aberrant methylation of the promoter region of 5'CpG island of BRCA1 gene. 3.1n the 5 cases of healthy adult women's blood samples, no methylation of the promoter region of 5'CpG island of BRCA1 gene was detected. 4.Based on the...