BRCA1突变和异常表达与犬乳腺肿瘤相关性的分析

发布时间：2018-01-12 00:02

本文关键词：BRCA1突变和异常表达与犬乳腺肿瘤相关性的分析　出处：《中国农业大学》2016年博士论文　论文类型：学位论文

【摘要】：乳腺癌易感基因1(Breast cancer 1,early onset, BRCAI)突变是导致人家族型乳腺癌发生的主要原因之一。但是关于犬BRCA1在乳腺肿瘤发生中的作用,研究尚不深入。在这篇论文中,检测了犬乳腺肿瘤中BRCA1启动子区的突变情况,mRNA和蛋白的表达情况,编码区的突变情况和BRCA1启动子区的甲基化情况。并希望通过这些实验,进一步阐明BRCA1在犬乳腺肿瘤发生中的作用。由于犬BRCA1启动子序列尚没有确定,首先通过生物信息学的方法预测了犬BRCA1基因的启动子序列,随后用启动子活性分析的方法进行验证。研究结果显示,预测的犬BRCA1启动子组的报告基因活性明显高于两个阴性对照组(P0.01)。为了检测犬乳腺肿瘤中BRCA1启动子区的突变情况,从中国农业大学动物医院获取犬乳腺肿瘤组织样本,根据病理特征将所得的样本分成良性乳腺肿瘤组和恶性乳腺肿瘤组,每组均有15例组织样本,并川10例犬止常乳腺组织作为对照组。通过PCR产物测序,分析了各组组织中BRCA1启动子区的突变情况。启动子测序结果显示,在正常乳腺组织和良性乳腺肿瘤中,均没有检测到启动子区的突变；在恶性乳腺肿瘤中,在启动子区发现一个19959098Cdel的点突变,突变率为46.7%(7/15)。为了检测犬乳腺肿瘤中BRCA1表达的改变,分别通过定量PCR和免疫组织化学的方法,检测上述各组组织中BRCA1基因mRNA和蛋白的表达情况。研究结果显示,在犬的良性和恶性乳腺肿瘤中,BRCA1的mRNA表达量明显低于正常组织的对照组(P0.05)；BRCA1蛋白的表达量,恶性乳腺肿瘤组明显低于正常乳腺组织的对照组(P0.05)。为了检测犬乳腺肿瘤中BRCA1编码区的突变情况和启动子区的甲基化情况,分别用PCR产物测序和亚硫酸氢盐测序的方法,检测了上述各组组织中,BRCA1编码区的突变情况和启动子区的甲基化情况。研究结果显示,在正常乳腺组织中,没有检测到BRCA1启动子区发生突变；在乳腺肿瘤组织中,检测到BRCA1编码区的4个突变位点,其中3个为新发现的突变位点。在正常乳腺组织和良性乳腺肿瘤组织DNA样本中,均没有检测到BRCA1启动子区的甲基化；在1例恶性乳腺乳腺肿瘤组织DNA样本中,检测到BRCA1启动子区发生甲基化。综上所述,在犬恶性乳腺肿瘤中,检测到位于BRCA1启动子区的一个高突变率点突变位点。在犬良性和恶性乳腺肿瘤中,BRCA1 mRNA的水平有明显的下调。在犬恶性乳腺肿瘤中,BRCA1蛋白的水平有明显的下调。在犬的乳腺肿瘤中,检测到位于编码区的3个新的点突变位点。在犬的恶性乳腺肿瘤中,检测到BRCA1启动子区发生甲基化。
[Abstract]:Breast cancer susceptibility gene 1 (Breast cancer 1, early onset, BRCAI) mutation is a major cause of human familial breast cancer. But on the role of canine BRCA1 in breast tumors, has not been studied in detail. In this paper, the detection of mutations in BRCA1 promoter region of canine breast tumor the expression of mRNA and protein, encoding region mutation and hypermethylation of the BRCA1 promoter region. And hope that through these experiments, to further elucidate the role of BRCA1 in canine mammary tumors. The canine BRCA1 promoter sequence has not been established, first by bioinformatics methods to predict promoter the sequence of canine BRCA1 gene, then analysis method with promoter activity was verified. The results showed that the prediction of canine BRCA1 promoter reporter gene activity sub group was significantly higher than two of the negative control group (P0.01) for detection. Mutations in the promoter region of BRCA1 canine breast tumor, canine breast tumor tissue samples obtained from the China Agricultural University animal hospital, according to the pathological features of the samples are divided into benign breast tumor group and malignant breast tumor group, each group had 15 cases of tissue samples, and 10 cases of normal canine Sichuan breast tissue as the control group. The PCR products sequencing analysis, mutation of BRCA1 promoter region were tissues. Promoter sequencing results showed that in normal breast tissues and benign breast tumors, were not detected mutations in the promoter region; in malignant breast tumor, found in the promoter region of a 19959098Cdel point mutation, the mutation rate was 46.7% (7/15). In order to detect the expression of BRCA1 in canine mammary tumor changes respectively by the method of quantitative PCR and immunohistochemistry, the expression of BRCA1 gene and mRNA protein was detected in the two groups . the results showed that the dogs in benign and malignant breast tumors, BRCA1 expression of mRNA in the control group were significantly lower than that in normal tissue (P0.05); the expression of BRCA1, malignant breast tumor group was significantly lower than the control group of normal breast tissue (P0.05). The methylation status of BRCA1 encoding region for detection of canine mammary gland tumor mutation and promoter, respectively by PCR sequencing and bisulfite sequencing, measured in the two groups, the methylation status of BRCA1 encoding region mutation and promoter region. The results showed that in normal breast tissues, BRCA1 was not detected in the promoter region mutation; in breast cancer, detected 4 mutations of BRCA1 encoding region, of which 3 was a new mutation. In normal breast tissues and benign breast tumor tissue samples of DNA, were not detected by BRCA1 The promoter methylation in DNA samples; 1 cases of malignant breast tumor tissues, to detect BRCA1 methylation in promoter region. To sum up, in canine malignant mammary tumors, detected a BRCA1 located in the promoter region of the high mutation rate of point mutation. In benign and malignant canine breast tumor BRCA1, the level of mRNA was down regulated. In canine malignant breast tumors, BRCA1 protein level was down regulated. In canine breast tumor detection, 3 new points are located in the encoding region of the mutation. In malignant breast tumors in dogs, to detect BRCA1 methylation in promoter region.

【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S858.292

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