【作者】 任婕；

【导师】 金万宝；

【作者基本信息】 中国医科大学 ， 药理学， 2006， 博士

【摘要】 目的乳腺癌是严重威胁女性健康的恶性肿瘤之一，乳腺癌分为遗传型和散发型，遗传型约占5％，大部分(95％)是散发性乳腺癌。而80％家族遗传性乳腺癌和卵巢癌患者携带乳腺癌易感基因BRCA1突变。在散发性乳腺癌组织中很少检测到BRCA1基因突变，但有BRCA1 mRNA水平的降低和蛋白低表达，其机制还不清楚。随着对表观遗传机制的认识，有研究认为BRCA1基因启动子甲基化是导致BRCA1蛋白低表达的原因之一。本研究拟检测较大样本的乳腺组织中，乳腺癌易感基因BRCA1启动子CpG区甲基化状态，结合BRCA1蛋白的表达情况，探讨乳腺癌易感基因BRCA1在散发性乳腺癌中沉默的机制；检测BRCA1基因在非癌性乳腺组织、BRCA1启动子甲基化和非甲基化的乳腺癌组织中的拷贝数，探讨BRCA1基因启动子甲基化与BRCA1基因位点缺失的关系，散发性乳腺癌的发病机制。方法免疫组织化学(immunohistochemistry，IHC)SP法检测98例散发性乳腺癌和12例非癌性乳腺组织石蜡标本BRCA1蛋白的表达情况。并分析BRCA1蛋白表达与临床病理学参数(患者年龄、乳腺癌病理类型、病理组织学分级、淋巴结的转移、雌、孕激素受体水平)，及与其他抑癌基因和癌基因(p53、C-myc、bcl-2和HER-2)蛋白表达之间的关系。甲基化特异性PCR技术(methylation specific PCR，MSP)检测98例乳腺癌和12例非癌性乳腺组织的冰冻标本BRCA1启动子区CpG岛甲基化水平，结合BRCA1蛋白表达情况，分析BRCA1启动子甲基化与BRCA1蛋白表达、临床病理指标和相关调控蛋白表达间的关系。采用间期核双色荧光免疫原位杂交(fluorescence in situ hybridization，FISH)技术，检测在非癌性乳腺组织、BRCA1基因启动子甲基化和非BRCA1甲基化的散发性乳腺癌组织中BRCA1基因和17号染色体着丝粒(CEP17)在17号染色体的拷贝数，计算拷贝的平均值和BRCA1／CEP17的比率，分析BRCA1启动子甲基化与BRCA1基因拷贝数之间的关系。结果一、BRCA1蛋白在乳腺组织中的表达：免疫组织化学检测结果表明，BRCA1蛋白在非癌性乳腺组织中的细胞核阳性表达率为100％(12／12)，未见细胞浆表达；在散发性乳腺癌组织中，BRCA1在细胞浆和细胞核均见表达，BRCA1在细胞核的阳性表达率为53.1％(52／98)。二、散发性乳腺癌组织中BRCA1表达与临床病理指标的相关性：免疫组化学检测显示，乳腺癌组织学分级Ⅰ级为29例(29.6％)，Ⅱ级为42例(42.9％)，Ⅲ级为27例(27.5％)。BRCA1蛋白表达阳性率在组织学分级Ⅰ、Ⅱ、Ⅲ中分别为72.4％(21／29)、50.0％(21／42)和37.0％(10／27)；其中BRCA1蛋白中度表达，在组织学分级Ⅰ、Ⅱ、Ⅲ中分别占27.6％(8／29)、4.8％(2／42)和0.0％(0／27)；低度表达在各组织分级比率分别为44.8％(13／29)、45.2％(19／42)和37.0％(10／27)；BRCA1蛋白表达阴性率分别为27.6％(8／29)、50.0％(21／42)和63.0％(17／27)。组织学分级Ⅰ、Ⅱ、Ⅲ中BRCA1蛋白阳性表达在细胞核的比率分别为55.2％(16／29)、40.5％(17／42)和11.1％(3／27)。BRCA1蛋白阳性表达率与组织学分级呈负相关(P＜0.01)，组织学分级越高，BRCA1蛋白表达越少。病例中腋淋巴结转移45例(45.9％)，无转移53例(54.1％)。BRCA1蛋白中度表达的腋淋巴结转移率10％(1／10)，低度表达的转移率38.1％(16／42)，BRCA1蛋白表达阴性的腋淋巴结转移率为60.9％(28／46)。BRCA1蛋白在细胞核阳性表达伴有腋淋巴结转移的为19.4％(7／36)，无淋巴结转移的为80.6％(29／36)。BRCA1蛋白细胞核阳性表达与腋淋巴结转移呈负相关(P＜0.01)。BRCA1蛋白表达与散发性乳腺癌雌激素受体(ER)和孕激素受体(PR)蛋白表达之间无显著相关性。三、散发性乳腺癌组织中BRCA1表达与相关调控基因蛋白表达的相关性：HER-2低表达的乳腺癌BRCA1细胞核表达阳性率为45.2％(14／31)，HER-2中、高度表达的乳腺癌组织BRCA1细胞核表达阳性率为11.1％(1／9)和0.0％(0／5)，BRCA1细胞核表达阴性的乳腺癌组织HER-2表达增高(P＜0.05)。其他相关调控蛋白(p53、bcl-2和C-myc)在BRCA1表达阴性的乳腺癌组织中，阳性表达率普遍高于BRCA1中度表达的乳腺癌组织，但无差异显著性。四、乳腺组织中BRCA1启动子甲基化检测：应用MSP方法检测了12例非癌性乳腺组织和98例散发性乳腺癌组织BRCA1基因启动子甲基化水平，在12例非癌性乳腺组织中，未检测到BRCA1启动子CpG区甲基化；在98例散发性乳腺癌组织中，检测到15例BRCA1启动子CpG区异常甲基化，占所检测散发性乳腺癌病例的15.3％(15／98)。五、BRCA1启动子甲基化与BRCA1蛋白及相关调控基因蛋白表达的相关性：MSP检测的98例散发性乳腺癌中，15例BRCA1启动子CpG区异常甲基化，结合免疫组化结果，该15例的细胞核BRCA1蛋白表达均为阴性，占BRCA1蛋白表达阴性病例的32.6％(15／46)。BRCA1启动子甲基化与BRCA1蛋白细胞核阴性表达密切相关(P＜0.01)。BRCA1启动子甲基化与相关调控蛋白bcl-2、p53、HER-2和c-myc表达之间，未见明显相关性。六、BRCA1基因启动子甲基化与乳腺癌临床病理指标的相关性：病理组织学分级Ⅰ级的乳腺癌组织，3.4％的病例检测到BRCA1启动子甲基化；而病理组织学分级Ⅲ级的乳腺癌，29.6％的病例检测到BRCA1启动子甲基化，差异性显著(P＜0.05)。淋巴结转移的散发性乳腺癌组织，24.4％(11／45)的病例BRCA1启动子甲基化，明显高于无淋巴结转移的乳腺癌组织中7.5％(4／53)的BRCA1启动子甲基化比率，差异性显著(P＜0.05)。七、BRCCA1拷贝数与BRCA1启动子甲基化的相关性：双色FISH检测非癌性乳腺组织3例、BRCA1启动子甲基化乳腺癌12和非甲基化的散发性乳腺癌组织5例的BRCA1基因拷贝，计数均值和BRCA1单拷贝和多拷贝所占比例。非癌性乳腺组织中，BRCA1基因平均拷贝数为2.05±0.09；BRCA1启动子甲基化的乳腺癌BRCA1基因平均拷贝数为1.41±0.18，低于非甲基化的散发性乳腺癌组织的BRCA1平均拷贝数1.98±0.09，差异显著(P＜0.05)。BRCA1启动子甲基化的乳腺癌BRCA1单拷贝比例为(22.04±1.06)％，高于BRCA1启动子非甲基化乳腺癌BRCA1单拷贝比例(2.56±0.32)％、非癌性乳腺组织中BRCA1单拷贝比率(0.84±0.03)％，差异性显著(P＜0.01)。BRCA1启动子非甲基化乳腺癌BRCA1基因多拷贝间期核的比例为(20.86±1.92)％，明显高于BRCA1启动子甲基化乳腺癌中(1.47±0.20)％的比例、非癌性乳腺组织中BRCA1多拷贝比率(1.12±0.08)％之间，有显著性差异(P＜0.01)。BRCA1启动子甲基化乳腺癌BRCA1基因平均拷贝数减少，单拷贝比率增加。八、BRCA1／CEP17拷贝数比率与BRCA1启动子甲基化的相关性：双色FISH检测乳腺非癌性组织、BRCA1启动子甲基化和非甲基化的散发性乳腺癌组织中CEP17拷贝，计算平均值和BRCA1／CEP17的平均拷贝比率。非癌性乳腺组织中，CEP17的平均拷贝数为2.33±0.17；BRCA1启动子甲基化的散发性乳腺癌组织中CEP17平均拷贝数为1.92±0.19，低于非甲基化乳腺癌中平均拷贝数2.39±0.46，但没有差异显著性。BRCA1启动子甲基化的乳腺癌CEP17单拷贝的比例为(24.60±2.09)％，高于BRCA1启动子非甲基化乳腺癌CEP17单拷贝的比例(3.31±0.23)％、非癌性乳腺组织中CEP17单拷贝比率(1.04±0.07)％，差异性显著(P＜0.01)。BRCA1启动子非甲基化乳腺癌CEP17多拷贝间期核的比例为(23.2±1.98)％，明显高于BRCA1启动子甲基化乳腺癌中(2.02±0.22)％的比例，非癌性乳腺组织中CEP17多拷贝比率(1.46±0.11)％，有显著性差异(P＜0.01)。BRCA1／CEP17比率在BRCA1启动子甲基化乳腺癌中平均为0.74±0.11，与非甲基化乳腺癌中的平均值0.85±0.13、非癌性乳腺组织中BRCA1／CEP17比率的均值0.88±0.05之间，无差异显著性。结论一、BRCA1蛋白在非癌性乳腺组织中均为细胞核阳性表达；在散发性乳腺癌组织中细胞核阳性表达下降，且与散发性乳腺癌的病理组织学分级、腋淋巴结转移、HER-2异常扩增等指标呈负相关。‘二、散发性乳腺癌组织中，BRCA1启动子甲基化与蛋白低表达密切相关，可能机制为BRCA1启动子甲基化导致BRCA1和CEP17拷贝缺失所致。更多还原

【Abstract】 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.更多还原