本文選題：甲基化　切入點(diǎn)：BSP　出處：《河北醫(yī)科大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：在中國(guó),乳腺癌已位居于女性惡性腫瘤發(fā)病首位,嚴(yán)重威脅著中國(guó)女性的健康。在臨床上,三陰性乳腺癌(Triple negative breast cancer,TNBC)由于其雌激素受體(estrogen receptor,ER)、孕激素受體(progesterone receptor,PR)、及人表皮生長(zhǎng)因子2(human epidermal receptor 2,Her2)表達(dá)均為陰性,因此對(duì)于內(nèi)分泌治療和分子靶向藥物赫賽汀治療無(wú)效,缺乏針對(duì)性的有效治療。TNBC的治療目前是乳腺癌治療領(lǐng)域的熱點(diǎn)及難點(diǎn)之一。乳腺癌易感基因1(breast cancer susceptibility gene l,BRCA1)是與乳腺癌密切相關(guān)的抑癌基因。BRCA1的缺失可導(dǎo)致乳腺癌的發(fā)生發(fā)展。大量研究證實(shí)BRCA1基因突變,抑制BRCA1基因表達(dá),從而導(dǎo)致乳腺癌的發(fā)生發(fā)展,且與家族性乳腺癌密切相關(guān)。但在散發(fā)性乳腺癌中,BRCA1基因的序列是完整的,卻檢測(cè)到其m RNA和蛋白表達(dá)水平明顯減少,說(shuō)明影響B(tài)RCA1基因表達(dá)水平的機(jī)制,除BRCA1基因突變,還有其他機(jī)制導(dǎo)致BRCA1基因表達(dá)減低。目前的研究認(rèn)為基因異常甲基化在正常細(xì)胞發(fā)生惡性轉(zhuǎn)變以及在惡性腫瘤的侵襲性逐漸增強(qiáng)的過(guò)程中占有重要的作用。且有研究發(fā)現(xiàn)TNBC中BRCA1基因甲基化率較高。目的:探討三陰性乳腺癌中乳腺癌易感基因BRCA1啟動(dòng)子和第一外顯子異常甲基化與其表達(dá)之間的關(guān)系。進(jìn)而為三陰性乳腺癌的治療開(kāi)辟新思路。方法:1應(yīng)用免疫組織化學(xué)方法檢測(cè)不同分子分型的乳腺癌組織中BRCA1蛋白的表達(dá)。2應(yīng)用實(shí)時(shí)熒光定量PCR法(Real-time Quantitative PCR,RT-q PCR)檢測(cè)不同分子分型乳腺癌細(xì)胞中BRCA1 m RNA表達(dá)水平。3應(yīng)用實(shí)時(shí)熒光定量PCR法(Real-time Quantitative PCR,RT-q PCR)檢測(cè)不同分子分型乳腺癌細(xì)胞中藥物對(duì)BRCA1 m RNA表達(dá)的影響。4應(yīng)用免疫細(xì)胞化學(xué)方法檢測(cè)不同分子分型乳腺癌細(xì)胞中BRCA1蛋白的表達(dá)水平。5應(yīng)用亞硫酸氫鹽測(cè)序法(Bisulfite sequencing PCR,BSP)分別檢測(cè)不同分子分型乳腺癌細(xì)胞中BRCA1啟動(dòng)子和第一外顯子Cp G島甲基化水平。結(jié)果:1不同分子分型的乳腺癌組織中BRCA1蛋白表達(dá)水平組織芯片免疫組織化學(xué)結(jié)果顯示(見(jiàn)圖1、2)(見(jiàn)表1):TNBC BRCA1蛋白表達(dá)率57.9%(11/19);Luminal型乳腺癌BRCA1蛋白表達(dá)率85.5%(59/69)和HER2+型乳腺癌BRCA1蛋白表達(dá)率76.9%(10/13)。TNBC組BRCA1蛋白表達(dá)率低于Luminal組有統(tǒng)計(jì)學(xué)意義(P0.05);TNBC組BRCA1蛋白表達(dá)率低于HER2+組,但差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05);HER2+型組BRCA1蛋白表達(dá)率低于Luminal型組,但差異無(wú)統(tǒng)計(jì)學(xué)意義(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結(jié)果顯示(見(jiàn)圖3)(見(jiàn)表2):三種分型乳腺癌患者總生存有差異(P0.05)。其中TNBC組和HER2+型組患者與Luminal組相比總生存期明顯縮短,有統(tǒng)計(jì)學(xué)差異(P0.05);TNBC組患者與HER2+型組相比總生存期無(wú)統(tǒng)計(jì)學(xué)差異(P0.05)。3 BRCA1蛋白表達(dá)陽(yáng)性和陰性乳腺癌患者的生存分析BRCA1蛋白表達(dá)陽(yáng)性和陰性乳腺癌患者的生存分析結(jié)果顯示(見(jiàn)圖4)(見(jiàn)表2):BRCA1蛋白表達(dá)陰性患者與BRCA1蛋白表達(dá)陽(yáng)性患者相比總生存期縮短,差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。4不同分子分型的乳腺癌細(xì)胞中BRCA1 m RNA的表達(dá)水平應(yīng)用實(shí)時(shí)熒光定量PCR(RT-q PCR)檢測(cè)乳腺癌細(xì)胞MDA-MB-231、BT-549、MCF-7、MDA-MB-453中BRCA1 m RNA表達(dá)水平。BRCA1m RNA的表達(dá)水平通常用2-△△CT值來(lái)表示。結(jié)果顯示(見(jiàn)圖5):MDA-MB-231、BT549、MCF-7、MDA-MB-453細(xì)胞中BRCA1 m RNA表達(dá)水平分別為1.00±0.08、1.11±0.14、2.42±0.21、5.89±0.65。三陰性乳腺癌細(xì)胞MDA-MB-231與TNBC細(xì)胞BT549 BRCA1 m RNA表達(dá)無(wú)統(tǒng)計(jì)學(xué)差異;TNBC細(xì)胞MDA-MB-231和BT549 BRCA1 m RNA表達(dá)均小于Luminal型乳腺癌細(xì)胞MCF-7和HER2+型乳腺癌細(xì)胞MDA-MB-453,有統(tǒng)計(jì)學(xué)意義(P0.001);Luminal型乳腺癌細(xì)胞MCF-7細(xì)胞BRCA1 m RNA的表達(dá)小于HER2+型乳腺癌細(xì)胞MDA-MB-453,有統(tǒng)計(jì)學(xué)意義(P0.001)。5去甲基化藥物(5-Aza-DC)對(duì)不同分子分型的乳腺癌細(xì)胞BRCA1m RNA表達(dá)水平的影響。乳腺癌細(xì)胞MDA-MB-231、BT-549、MCF-7、MDA-MB-453分別經(jīng)1mmol/L 5-Aza-DC、2mmol/L 5-Aza-DC處理72h后,通過(guò)RT-q PCR檢測(cè)BRCA1 m RNA表達(dá)水平。結(jié)果顯示(見(jiàn)圖6):MDA-MB-231細(xì)胞:1mmol/L 5-Aza-DC處理組BRCA1 m RNA的表達(dá)水平為(1.94±0.24),2mmol/L 5-Aza-DC處理組BRCA1 m RNA的表達(dá)水平為(2.35±0.66),均與未加藥組(1.00±0.08)相比顯著增高,差異均具有統(tǒng)計(jì)學(xué)意義(P0.05)。BT-549細(xì)胞:1mmol/L 5-Aza-DC處理組BRCA1 m RNA的表達(dá)水平(1.22±0.08)與未加藥組(1.01±0.13)差別無(wú)統(tǒng)計(jì)學(xué)意義,2mmol/L5-Aza-DC處理組BRCA1 m RNA的表達(dá)水平(2.69±0.34)與未加藥組相比顯著增高,差異有統(tǒng)計(jì)學(xué)意義(P0.01)。MCF-7細(xì)胞:1mmol/L 5-Aza-DC處理組BRCA1 m RNA的表達(dá)水平(1.15±0.19)與未加藥組(1.00±0.09)差別無(wú)統(tǒng)計(jì)學(xué)意義,2mmol/L5-Aza-DC處理組BRCA1 m RNA的表達(dá)水平(1.42±0.18)與未加藥組相比顯著增高,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。MDA-MB-453細(xì)胞:1mmol/L 5-Aza-DC處理組BRCA1 m RNA的表達(dá)水平為(3.18±0.70),2mmol/L 5-Aza-DC處理組BRCA1 m RNA的表達(dá)水平為(5.97±0.87),均與未加藥組(1.00±0.11)相比顯著增高,差異均具有統(tǒng)計(jì)學(xué)意義(P0.01)。6不同分子分型的乳腺癌細(xì)胞中BRCA1蛋白的表達(dá)水平本實(shí)驗(yàn)應(yīng)用免疫細(xì)胞化學(xué)的方法檢測(cè)四種乳腺癌細(xì)胞中BRCA1蛋白水平。BRCA1蛋白免疫細(xì)胞化學(xué)陽(yáng)性信號(hào)均呈棕黃色,即存在于細(xì)胞質(zhì)中也存在于細(xì)胞核中。結(jié)果顯示(見(jiàn)圖7):TNBC細(xì)胞MDA-MB-231、BT549中BRCA1蛋白表達(dá)水平均低于Luminal型乳腺癌細(xì)胞MCF-7和HER2+型乳腺癌細(xì)胞MDA-MB-453。這與BRCA1 m RNA表達(dá)結(jié)果一致。7不同分子分型的乳腺癌細(xì)胞中BRCA1啟動(dòng)子甲基化率本實(shí)驗(yàn)應(yīng)用亞硫酸氫鹽測(cè)序法(BSP)檢測(cè)乳腺癌細(xì)胞BRCA1啟動(dòng)子(-908~-714)甲基化率。結(jié)果顯示(見(jiàn)圖14、15):MDA-MB-231、BT-549、MCF-7、MDA-MB-453細(xì)胞系甲基化率分別是(26/180)14.44%、(7/180)3.89%、(4/180)2.22%、(2/180)1.11%。與BRCA1 m RNA、蛋白表達(dá)負(fù)相關(guān)。8不同分子分型的乳腺癌細(xì)胞中BRCA1第一外顯子甲基化率本實(shí)驗(yàn)應(yīng)用亞硫酸氫鹽測(cè)序法(BSP)檢測(cè)乳腺癌細(xì)胞BRCA1第一外顯子(-70~165)甲基化率。結(jié)果顯示(見(jiàn)圖14、15):MDA-MB-231、BT-549、MCF-7、MDA-MB-453細(xì)胞系甲基化率分別是(6/182)3.30%、(1/182)0.55%、(13/182)7.14%、(0/180)0.00%。與BRCA1 m RNA、蛋白表達(dá)無(wú)相關(guān)性。結(jié)論:1三陰性乳腺癌中BRCA1基因表達(dá)較Luminal型和HER2+型表達(dá)低,且與BRCA1啟動(dòng)子(-908~-714)的甲基化率呈負(fù)相關(guān)。2三陰性乳腺癌中BRCA1第一外顯子(-70~165)的甲基化率與BRCA1基因表達(dá)無(wú)關(guān)。
[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.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R737.9

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