三阴性乳腺癌中BRCA1基因启动子和外显子异常甲基化与其表达的分析

发布时间：2018-03-21 21:48

本文选题：甲基化　切入点：BSP　出处：《河北医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：在中国,乳腺癌已位居于女性恶性肿瘤发病首位,严重威胁着中国女性的健康。在临床上,三阴性乳腺癌(Triple negative breast cancer,TNBC)由于其雌激素受体(estrogen receptor,ER)、孕激素受体(progesterone receptor,PR)、及人表皮生长因子2(human epidermal receptor 2,Her2)表达均为阴性,因此对于内分泌治疗和分子靶向药物赫赛汀治疗无效,缺乏针对性的有效治疗。TNBC的治疗目前是乳腺癌治疗领域的热点及难点之一。乳腺癌易感基因1(breast cancer susceptibility gene l,BRCA1)是与乳腺癌密切相关的抑癌基因。BRCA1的缺失可导致乳腺癌的发生发展。大量研究证实BRCA1基因突变,抑制BRCA1基因表达,从而导致乳腺癌的发生发展,且与家族性乳腺癌密切相关。但在散发性乳腺癌中,BRCA1基因的序列是完整的,却检测到其m RNA和蛋白表达水平明显减少,说明影响BRCA1基因表达水平的机制,除BRCA1基因突变,还有其他机制导致BRCA1基因表达减低。目前的研究认为基因异常甲基化在正常细胞发生恶性转变以及在恶性肿瘤的侵袭性逐渐增强的过程中占有重要的作用。且有研究发现TNBC中BRCA1基因甲基化率较高。目的:探讨三阴性乳腺癌中乳腺癌易感基因BRCA1启动子和第一外显子异常甲基化与其表达之间的关系。进而为三阴性乳腺癌的治疗开辟新思路。方法:1应用免疫组织化学方法检测不同分子分型的乳腺癌组织中BRCA1蛋白的表达。2应用实时荧光定量PCR法(Real-time Quantitative PCR,RT-q PCR)检测不同分子分型乳腺癌细胞中BRCA1 m RNA表达水平。3应用实时荧光定量PCR法(Real-time Quantitative PCR,RT-q PCR)检测不同分子分型乳腺癌细胞中药物对BRCA1 m RNA表达的影响。4应用免疫细胞化学方法检测不同分子分型乳腺癌细胞中BRCA1蛋白的表达水平。5应用亚硫酸氢盐测序法(Bisulfite sequencing PCR,BSP)分别检测不同分子分型乳腺癌细胞中BRCA1启动子和第一外显子Cp G岛甲基化水平。结果:1不同分子分型的乳腺癌组织中BRCA1蛋白表达水平组织芯片免疫组织化学结果显示(见图1、2)(见表1):TNBC BRCA1蛋白表达率57.9%(11/19);Luminal型乳腺癌BRCA1蛋白表达率85.5%(59/69)和HER2+型乳腺癌BRCA1蛋白表达率76.9%(10/13)。TNBC组BRCA1蛋白表达率低于Luminal组有统计学意义(P0.05);TNBC组BRCA1蛋白表达率低于HER2+组,但差异无统计学意义(P0.05);HER2+型组BRCA1蛋白表达率低于Luminal型组,但差异无统计学意义(P0.05)。2不同分子分型乳腺癌患者的生存分析不同分子分型的乳腺癌患者生存分析结果显示(见图3)(见表2):三种分型乳腺癌患者总生存有差异(P0.05)。其中TNBC组和HER2+型组患者与Luminal组相比总生存期明显缩短,有统计学差异(P0.05);TNBC组患者与HER2+型组相比总生存期无统计学差异(P0.05)。3 BRCA1蛋白表达阳性和阴性乳腺癌患者的生存分析BRCA1蛋白表达阳性和阴性乳腺癌患者的生存分析结果显示(见图4)(见表2):BRCA1蛋白表达阴性患者与BRCA1蛋白表达阳性患者相比总生存期缩短,差异无统计学意义(P0.05)。4不同分子分型的乳腺癌细胞中BRCA1 m RNA的表达水平应用实时荧光定量PCR(RT-q PCR)检测乳腺癌细胞MDA-MB-231、BT-549、MCF-7、MDA-MB-453中BRCA1 m RNA表达水平。BRCA1m RNA的表达水平通常用2-△△CT值来表示。结果显示(见图5):MDA-MB-231、BT549、MCF-7、MDA-MB-453细胞中BRCA1 m RNA表达水平分别为1.00±0.08、1.11±0.14、2.42±0.21、5.89±0.65。三阴性乳腺癌细胞MDA-MB-231与TNBC细胞BT549 BRCA1 m RNA表达无统计学差异;TNBC细胞MDA-MB-231和BT549 BRCA1 m RNA表达均小于Luminal型乳腺癌细胞MCF-7和HER2+型乳腺癌细胞MDA-MB-453,有统计学意义(P0.001);Luminal型乳腺癌细胞MCF-7细胞BRCA1 m RNA的表达小于HER2+型乳腺癌细胞MDA-MB-453,有统计学意义(P0.001)。5去甲基化药物(5-Aza-DC)对不同分子分型的乳腺癌细胞BRCA1m RNA表达水平的影响。乳腺癌细胞MDA-MB-231、BT-549、MCF-7、MDA-MB-453分别经1mmol/L 5-Aza-DC、2mmol/L 5-Aza-DC处理72h后,通过RT-q PCR检测BRCA1 m RNA表达水平。结果显示(见图6):MDA-MB-231细胞:1mmol/L 5-Aza-DC处理组BRCA1 m RNA的表达水平为(1.94±0.24),2mmol/L 5-Aza-DC处理组BRCA1 m RNA的表达水平为(2.35±0.66),均与未加药组(1.00±0.08)相比显著增高,差异均具有统计学意义(P0.05)。BT-549细胞:1mmol/L 5-Aza-DC处理组BRCA1 m RNA的表达水平(1.22±0.08)与未加药组(1.01±0.13)差别无统计学意义,2mmol/L5-Aza-DC处理组BRCA1 m RNA的表达水平(2.69±0.34)与未加药组相比显著增高,差异有统计学意义(P0.01)。MCF-7细胞:1mmol/L 5-Aza-DC处理组BRCA1 m RNA的表达水平(1.15±0.19)与未加药组(1.00±0.09)差别无统计学意义,2mmol/L5-Aza-DC处理组BRCA1 m RNA的表达水平(1.42±0.18)与未加药组相比显著增高,差异有统计学意义(P0.05)。MDA-MB-453细胞:1mmol/L 5-Aza-DC处理组BRCA1 m RNA的表达水平为(3.18±0.70),2mmol/L 5-Aza-DC处理组BRCA1 m RNA的表达水平为(5.97±0.87),均与未加药组(1.00±0.11)相比显著增高,差异均具有统计学意义(P0.01)。6不同分子分型的乳腺癌细胞中BRCA1蛋白的表达水平本实验应用免疫细胞化学的方法检测四种乳腺癌细胞中BRCA1蛋白水平。BRCA1蛋白免疫细胞化学阳性信号均呈棕黄色,即存在于细胞质中也存在于细胞核中。结果显示(见图7):TNBC细胞MDA-MB-231、BT549中BRCA1蛋白表达水平均低于Luminal型乳腺癌细胞MCF-7和HER2+型乳腺癌细胞MDA-MB-453。这与BRCA1 m RNA表达结果一致。7不同分子分型的乳腺癌细胞中BRCA1启动子甲基化率本实验应用亚硫酸氢盐测序法(BSP)检测乳腺癌细胞BRCA1启动子(-908~-714)甲基化率。结果显示(见图14、15):MDA-MB-231、BT-549、MCF-7、MDA-MB-453细胞系甲基化率分别是(26/180)14.44%、(7/180)3.89%、(4/180)2.22%、(2/180)1.11%。与BRCA1 m RNA、蛋白表达负相关。8不同分子分型的乳腺癌细胞中BRCA1第一外显子甲基化率本实验应用亚硫酸氢盐测序法(BSP)检测乳腺癌细胞BRCA1第一外显子(-70~165)甲基化率。结果显示(见图14、15):MDA-MB-231、BT-549、MCF-7、MDA-MB-453细胞系甲基化率分别是(6/182)3.30%、(1/182)0.55%、(13/182)7.14%、(0/180)0.00%。与BRCA1 m RNA、蛋白表达无相关性。结论:1三阴性乳腺癌中BRCA1基因表达较Luminal型和HER2+型表达低,且与BRCA1启动子(-908~-714)的甲基化率呈负相关。2三阴性乳腺癌中BRCA1第一外显子(-70~165)的甲基化率与BRCA1基因表达无关。
[Abstract]:In Chinese, the incidence of breast cancer has ranked the first female malignant tumor, a serious threat to women's health China. Clinically, three negative breast cancer (Triple negative breast cancer, TNBC) because of its estrogen receptor (estrogen receptor, ER), progesterone receptor (progesterone receptor, PR), and human epidermal growth factor 2 (human epidermal receptor 2, Her2) expression were negative for endocrine therapy and molecular targeted drug therapy for treatment of Hessaitin, the lack of effective treatment for.TNBC is currently one of the hotspots and difficulties in breast cancer treatment field. Breast cancer susceptibility gene 1 (breast cancer susceptibility gene L, BRCA1) is breast cancer is closely associated with the deletion of tumor suppressor gene.BRCA1 can lead to the development of breast cancer. A large number of studies confirm that mutations in the BRCA1 gene, inhibit BRCA1 gene expression, which leads to the occurrence of breast cancer The development, which is closely related with familial breast cancer. But in sporadic breast cancer, BRCA1 gene sequence is complete, but the detected m RNA and protein expression level was significantly reduced, that influence the expression level of BRCA1 gene BRCA1 gene mutation mechanism, in addition, there are other mechanisms leading to reduced expression of BRCA1 gene at present. The study suggests that plays an important role in gene methylation in malignant transformation of normal cells and invasion in malignant tumor increased. And TNBC was found in the BRCA1 gene methylation rate is higher. Objective: To investigate the three negative breast cancer breast cancer susceptibility gene BRCA1 promoter and the first exon of the relationship between methylation and expression. And for the treatment of three negative breast cancer to open up new ideas. Methods: breast cancer of different molecular subtypes of 1 detected by immunohistochemical method in BR Real time fluorescence quantitative PCR expression of.2 protein (Real-time Quantitative CA1 PCR, RT-q PCR) to detect different molecular level of.3 by real-time fluorescent quantitative PCR method BRCA1 m RNA expression in breast cancer cells (Real-time, Quantitative PCR, RT-q PCR) to detect the effect of molecular drug type breast cancer cells on the expression of BRCA1 m RNA.4 immunocytochemical method with different molecular BRCA1 protein in breast cancer cells.5 expression using bisulfite sequencing method (Bisulfite sequencing PCR, BSP) were detected in different molecular BRCA1 promoter and the first type of breast cancer cells in exon Cp G island methylation level. Results: the level of tissue microarray immunohistochemistry showed that the expression of BRCA1 protein in 1 different molecular subtypes of breast cancer tissues (Figure 1,2) (see Table 1): the TNBC BRCA1 protein expression rate was 57.9% (11/ 19); type Luminal milk Adenocarcinoma the expression rate of BRCA1 was 76.9% and 85.5% (59/69) HER2+ type of breast cancer, the expression of BRCA1 protein (10/13) expression of.TNBC protein in BRCA1 group was lower than that in Luminal group were significant (P0.05); the expression of TNBC protein in BRCA1 group was lower than that of HER2+ group, but the difference was not statistically significant (P0.05); the expression of HER2+ protein in BRCA1 group the rate was lower than that of Luminal group, but the difference was not statistically significant (P0.05.2) of different molecular subtypes of breast cancer survival analysis of patients with breast cancer survival analysis results of different molecular subtypes showed (see Figure 3) (see Table 2): three types of breast cancer patients have differences in overall survival (P0.05) TNBC. Group and HER2+ group compared with Luminal group, the total survival period is shortened obviously, there was significant difference (P0.05); TNBC group and HER2+ group compared with no significant difference in overall survival (P0.05) positive and negative breast cancer survival analysis BRCA1 expression of.3 BRCA1 protein The expression of positive and negative results of survival analysis of patients with breast cancer shows (Figure 4) (see Table 2): Patients with negative and positive expression of BRCA1 protein in patients with shorter survival compared to the total BRCA1 protein expression, there was no statistically significant difference (P0.05) expression by real-time quantitative PCR.4 of different molecular subtypes of breast cancer cells BRCA1 m RNA (RT-q PCR) detection of breast cancer cells MDA-MB-231, BT-549, MCF-7,.BRCA1m expression level of RNA 2- is usually used to represent the value of delta CT BRCA1 m RNA expression in MDA-MB-453. The results showed (see Figure 5): MDA-MB-231, BT549, MCF-7, BRCA1 m expression level of RNA expression were 1 + 0.08,1.11 + 0.14,2.42 + 0.21,5.89 + 0.65. three negative breast cancer cells MDA-MB-231 and TNBC cells BT549 BRCA1 m RNA had no significant difference between MDA-MB-453 cells and BT549 cells; the expression of TNBC MDA-MB-231 BRCA1 m RNA are less than Luminal type breast The expression of MCF-7 and HER2+ in breast cancer cell line MDA-MB-453, with statistical significance (P0.001); the expression of Luminal in breast cancer cell line MCF-7 cells BRCA1 m RNA less than HER2+ type MDA-MB-453 breast cancer cells. There was statistical significance (P0.001).5 demethylation drugs (5-Aza-DC) on the expression of BRCA1m in breast cancer cells with RNA molecular typing of breast cancer cells. MDA-MB-231, BT-549, MCF-7, MDA-MB-453 respectively by 1mmol/L 5-Aza-DC, 2mmol/L 5-Aza-DC 72h, BRCA1 m RNA RT-q by measuring the level of PCR expression (see Figure 6). The results showed: MDA-MB-231 cells: the expression of 1mmol/L 5-Aza-DC BRCA1 m RNA for the treatment group (1.94 + 0.24), expression the level of 2mmol/L 5-Aza-DC BRCA1 m RNA for the treatment group (2.35 + 0.66), and no drug group (1 + 0.08) compared significantly increased, the differences were statistically significant (P0.05).BT-549 cells: 1mmol/L 5-Aza-DC treatment group BR The expression level of CA1 m RNA (1.22 + 0.08) and no drug group (1.01 + 0.13) there was no significant difference between the treatment groups, the expression level of 2mmol/L5-Aza-DC BRCA1 m RNA (2.69 + 0.34) compared with the untreated group increased significantly, the difference was statistically significant (P0.01) of.MCF-7 cells: the expression of 1mmol /L 5-Aza-DC treatment group BRCA1 m RNA (1.15 + 0.19) and no drug group (1 + 0.09) there was no significant difference between the treatment groups, the expression level of 2mmol/L5-Aza-DC BRCA1 m RNA (1.42 + 0.18) compared with the untreated group increased significantly, the difference was statistically significant (P0.05) of.MDA-MB-453 cells: the expression of 1mmol/L 5-Aza-DC BRCA1 m RNA treatment group for (3.18 + 0.70), the expression level of 2mmol/L 5-Aza-DC BRCA1 m RNA for the treatment group (5.97 + 0.87), and no drug group (1 + 0.11) compared significantly increased, the differences were statistically significant (P0.01.6) of different molecular subtypes of breast cancer cells Methods the expression of BRCA1 protein in this experiment using immunocytochemical detection of four kinds of BRCA1 protein levels in breast cancer cells.BRCA1 protein immunohistochemistry positive signal was brown, which exists in the cytoplasm or in the nucleus (see Figure 7). The results showed: TNBC cells MDA-MB-231, Luminal levels were lower than that of breast cancer MCF-7 cells and HER2+ breast cancer cell MDA-MB-453. and the BRCA1 m RNA expression of the breast cancer cells.7 of different molecular subtypes of BRCA1 promoter methylation rates of the experimental application of sulfurous acid hydrogen salt sequencing of BRCA1 protein expression in BT549 (BSP) detection of breast cancer cells BRCA1 promoter methylation rate (-908~-714) (see Figure 14,15). The results showed: MDA-MB-231, BT-549, MCF-7, MDA-MB-453 cell line methylation rate was 14.44% (26/180), (7/180) 3.89%, (4/180) 2.22%, 1.11%. (2 /180) and BRCA1 m RNA, protein expression Negative correlation between.8 of different molecular subtypes of breast cancer cells in the first exon of BRCA1 methylation rate in this experiment using bisulfite sequencing (BSP) detection of BRCA1 breast cancer cells first exon (-70~165) methylation rate (see Figure 14,15). The results showed: MDA-MB-231, BT-549, MCF-7, MDA-MB-453 cell lines the methylation rate was 3.30% (6/182), (1/182) 0.55%, (13/182) 7.14%, (0/180) 0.00%. BRCA1 and m RNA protein expression was not correlated. Conclusion: BRCA1 1 three negative breast cancer gene expression than the Luminal type and HER2+ type low expression, and BRCA1 promoter (-908~-714) methyl the rate of.2 was negatively related to three negative breast cancer in the first exon of BRCA1 (-70~165) and the methylation rate of BRCA1 gene expression.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9

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