Relationships Between Mutation And Altered Expression Of BRCA1 And Canine Mammary Tumors

Mutation of BRCAI is one of most important reasons leading to human familial breast cancer. However, the research is limited about the functions of BRCAI in canine mammary tumorigenesis. In the thesis, the mutation statuses of BRCAI promoter region in canine mammary tumors were tested; the expression levels of BRCAI mRNA and protein were detected and the mutation statuses of BRCA1 coding region and DNA methylation of BRCA1 promoter region were analyzed. The purpose of the experiments is to further elucidate the functions of BRCAI in canine mammary tumorigenesis.The promoter sequence of canine BRCAI had not identified. Therefore, bioinformatics analysis was used to predicted canine promoter sequence. The predicted canine promoter was confirmed by promoter activity analysis. The experiment result indicted that the promoter activity of the predicted promoter was significantly higher than two control groups (P<0.01). In order to analyze the mutation statuses of canine BRCAI promoter, the canine mammary tumor samples were acquired from the Veterinary Teaching Hospital of China Agricultural University and divided into benign mammary tumor group and malignant mammary tumor group based on pathological analysis. There were 15 samples in each of the two groups and 10 canine normal mammary tissues were used as control group. Through direct sequencing, the mutation statuses were analyzed. The sequencing result indicted that no mutation was detected in normal mammary tissue group and benign mammary tumor group. In malignant mammary tumor group, a "19959098Cdel" mutation was found with high ratio 46.7% (7/15)In order to analyze the alterations of BRCAI in canine mammary tumors, real time PCR and immunohistochemistry techniques were used to detect the expression levels of BRCAI mRNA and protein respectively. The result indicated that, the mRNA levels of BRCAI in canine benign and malignant mammary tumor groups were significantly lower than in normal mammary tissue group (P<0.05). The protein levels of BRCAI in canine malignant mammary tumor group were significantly lower than in normal mammary tissue group (P<0.05).In order to detect the mutation statuses of BRCAI coding region and DNA methylation of the promoter region, direct sequencing and busilfite sequencing were used. The results indicated that no mutation of BRCA1 coding region was detected in canine normal mammary tissue group. In the two canine mammary tumor groups, four mutation sites were detected in BRCAI coding region. Three of the four point mutations are newly discovered mutations. Busilfite sequencing results indicated that no DNA methylation was detected in normal and benign mammary tumor group. In canine malignant mammary tumor group, one sample was detected with DNA methylation.In conclusion, in canine BRCA1 promoter region, a mutation was found with high ratio in canine malignant mammary tumors. In canine benign and malignant mammary tumors, the mRNA levels were downregulated. In canine malignant mammary tumors, the protein levels were dowregulated. In canine mammary tumors, three new point mutations were found. In canine malignant mammary tumors, DNA methylation of the promoter region was found.