【作者】 陈杨萍；

【导师】 聂建云；

【作者基本信息】 昆明医科大学 ， 肿瘤学， 2012， 硕士

【摘要】 [背景和目的]乳腺癌是女性发病率较高的恶性肿瘤之一,全世界每年大概有1300000的乳腺癌新增病例,其中超过15%的患者是三阴性乳腺癌。三阴性乳腺癌(Triple-Negative Breast Cancer,TNBC)指的是雌激素受体(estrogen receptor,ER),孕激素受体(progesterone receptor,PR)和人表皮生长因子受体2(human epidermal growth factor receptor2,Her-2)均表达缺失的乳腺癌。此类乳腺癌一般组织学分级高、侵袭性强、预后差。对内分泌药物和针对Her-2的靶向治疗无效。相对于其他类型的乳腺癌,TNBC对含紫杉类和蒽环类的新辅助化疗有较高的病理完全缓解率和临床反应率,但是无复发生存率及总体生存率低于非TNBC。易在短期内复发,内脏转移率高于骨转移,脑转移率也较高,死亡风险高。这就使得为TNBC患者寻找一个有效地治疗药物成为一个亟待解决的问题。近年来,表观遗传学方面的研究逐渐受到人们的关注,表观遗传学是指不涉及DNA序列改变的基因或者蛋白表达的变化,并可以在发育和细胞增殖过程中稳定传递的遗传学。研究发现其与多种疾病、肿瘤的发生发展有关。组蛋白乙酰化修饰是表观遗传学中的重要组成部分,组蛋白乙酰化酶(HAT)和组蛋白去乙酰化酶(HDAC)之间的动态平衡控制着染色质结构和基因表达。两者间的动态平衡失调会导致肿瘤的发生。Ⅰ型HDAC中的HDAC1对肿瘤细胞的增殖起重要的的作用。HDAC抑制剂可通过调节异常增生和(或)凋亡基因的表达抑制肿瘤细胞的生长和存活。研究表明,HDAC抑制剂对多种肿瘤细胞,包括膀胱、卵巢、骨、食管、肺等均表现出良好的抗肿瘤作用。有研究证实HDAC抑制剂对雌激素受体阳性或阴性的乳腺癌细胞都有抑制作用,动物实验表明其与化疗药联用可增强其疗效,本实验构建HDAC1质粒,转染三阴性乳腺癌细胞(正向干预),另外将HDAC抑制剂作用与三阴性乳腺癌细胞(负向干预),通过观察细胞内周期素CyclinA2、CyclinD1和抑癌基因P21、14-3-3σ蛋白表达的变化探讨HDAC抑制剂对TNBC细胞增殖的影响机制,为临床上研发HDAC抑制剂做为一种新的治疗TNBC患者的分子靶向药物提供一个基础研究参考。[方法]本研究包括二部分1、组蛋白去乙酰化酶1对三阴性乳腺癌细胞增殖的影响(正向干预)1.1通过基因亚克隆(Subcloning)技术构建组蛋白去乙酰化酶1质粒(pcDNA3.1-hdacl)本实验室前期工作已完成；1.2CaCl2法制备感受态细菌(E.coli);1.3pcDNA3.1-hdacl质粒转化感受态细菌(E.coli)扩增质粒,进行质粒提纯、酶切鉴定；1.4pcDNA3.1-hdacl质粒转染三阴性乳腺癌细胞株,用Western印迹法检测质粒蛋白表达：1.5流式细胞术检测转染质粒前后细胞周期及四种蛋白Cyclin A2、Cyclin D1、P21、14-3-3σ的变化。1.6用Western印迹法检测四种蛋白Cyclin A2、Cyclin D1、P21、14-3-3σ的变化。2、组蛋白去乙酰化酶抑制剂对三阴性乳腺癌细胞增殖的影响(负向干预)2.1倒置显微镜下观察组蛋白去乙酰化酶抑制剂SAHA对三阴性乳腺癌细胞增殖的影响2.2MTS比色法计算组蛋白去乙酰化酶抑制剂SAHA对三阴性乳腺癌细胞增殖的影响；2.3流式细胞术检测组蛋白去乙酰化酶抑制剂SAHA对细胞周期和四种蛋白Cyclin A2、Cyclin D1、P21和14-3-3σ的影响。2.4用Western印迹法检测组蛋白去乙酰化酶抑制剂SAHA对四种蛋白Cyclin A2、Cyclin D1、P21和14-3-3σ的影响变化。[结果]1、组蛋白去乙酰化酶1对三阴性乳腺癌细胞增殖的影响(正向干预)1.1组蛋白去乙酰化酶1质粒(pcDNA3.1-hdac1)的构建与鉴定亚克隆构建组蛋白去乙酰化酶1质粒(pcDNA3.1-hdac1)成功后,进行质粒提纯、酶切鉴定,证实hdacl DNA片段为1449bp(克隆位点为BamH I/EcoR I);转染三阴性乳腺癌细胞株,Western印迹法检出有HDAC1蛋白表达1.2组蛋白去乙酰化酶1对三阴性乳腺癌细胞增殖周期的影响流式细胞术检测显示：未转染HDAC1质粒的三阴性乳腺癌细胞株MDA-MB-468细胞的G1,S期细胞的比例分别为(60.2±0.3)%,(13.7±0.2)%,而转染后的三阴性乳腺癌细胞株MDA-MB-468细胞中代表细胞增殖的S期细胞比例增加(36.1±0.3)%,P<0.05；G1期细胞比例下降(45.7-0.2)%,P<0.05。1.3组蛋白去乙酰化酶1对三阴性乳腺癌细胞中四种蛋白的影响流式细胞术检测显示：转染HDAC1后三阴性乳腺癌细胞的Cyclin A2较对照组增加,增幅超过20%,Cyclin D1、P21、14-3-3σ较对照组减少。Western印迹法亦表明转染组较对照组Cyclin A2增加,Cyclin D1、P21、14-3-3σ较对照组减少2、组蛋白去乙酰化酶抑制剂对三阴性乳腺癌细胞增殖的影响(负向干预)MTS比色法显示MDA-MB-468细胞在加入组蛋白酶去乙酰化酶抑制剂SAHA24h后出现细胞生长抑制,抑制效应随时间的增加而逐渐增加,并逐渐出现细胞凋亡,72h达高峰,FCM检测显示在加入SAHA48h后MDA-MB-468细胞出现S期细胞比例下降,细胞内CyclinA2蛋白的表达量下降,Cyclin D1、P21、14-3-3σ蛋白的表达量上升,细胞凋亡率上升,与MDA-MB-468对照组比较差异有统计学意义(P＜0.05), Western印迹法显示,MDA-MB-468细胞在加入SAHA48h后Cyclin A2蛋白表达量下降,Cycli0D1、P21、14-3-3σ蛋白的表达量上升。[结论]1、组蛋白去乙酰化酶1(HDAC1)基因在三阴性乳腺癌细胞中有表达,其表达对三阴性乳腺癌细胞的增殖有促进作用。2、SAHA是TNBC细胞增殖的高效抑制剂并促进其凋亡,其抑制作用有一定的时效关系：抑癌基因P21、14-3-3σ和周期数Cyclin A2、cyclinD1参与SAHA对细胞增殖周期的调控。更多还原

【Abstract】 [Background and Objective]Breast cancer is one of the higher mortality rates of malignant tumors for woman. There are about1,300,000new cases of breast cancer all over the world. Among these new patients, there are over15%are triple negative breast cancer. Breast cancer, which show estrogen receptor(ER), progesterone receptor (PR) and human epidermal growth factor receptor2(Her-2) all negative, is called tripe negative breast cancer (TNBC). This kind of breast tumor always has higher histologic grade, stronger invasion and poor prognosis. It is invalid to endocrine drugs and targeted therapy drugs for Her-2. Compared to other kinds of breast cancer, patience of TNBC can get higher pathologic complete remission and clinical response rates to the neoadjuvant chemotherapy which contains paclitaxel and anthracycline, but also would get lower recurrence-free and overall survival rates. It has an easier shorten recurrence, a higher rate of brain metastases and internal metastases than bone metastases, and also a higher death rate. So it becomes an urgent problem to find an effective therapeutic drug for TNBC patients.In recent years, people gradually pay attention to the research on Epigenetics. Epigenetics, which is a branch of genetics and can transfer stably during development and cell proliferation, is not related to DNA sequence alterations of gene or protein expression changes. It is found that epigenetics related to the occurrence and development of many diseases and cancers. Histone acetylation is an important part of epigenetics. The dynamic equilibrium between Histone acetyltransferase (HAT) and histone deacetylase (HDAC) can control the chromatin structure and gene expression. The disorders of their dynamic balance can lead to tumor. HDAC1, type I of HDAC, play an important role on the proliferation of tumor cell. HDAC inhibitors can inhibit the growth and survival of tumor cell through regulating the abnormal proliferation and (or) the expression of apoptosis-related gene. Research shows that HDAC inhibitors have shown a good antitumor effect on a variety of tumor cells including bladder, ovarian, lung, bone, esophageal. Studies have confirmed that HDAC inhibitors can inhibit estrogen receptor positive or negative breast cancer cells. The animal experiment shows that the combination between HDAC inhibitor and chemotherapeutic drugs may enhance the curative effect. Our experiment is to make the construction of HDAC1plasmid and transfer it to the TNBC cells (positive intervention). Meanwhile we put HDAC inhibitor on the other TNBC cells and look for the mechanism by observing the changes of expression on cell cyclin cyclinA2, cyclin D2and tumor suppressor gene P21,14-3-3sigma protein. It can provide a basic study reference for developing HDAC inhibitor as a new molecular targeted drug for TNBC patients.【Methods】 The study includes two parts1. Effect of histone deacetylase1(HDAC1) on triple negative breast cancer cell proliferation (Positive intervention)1.1To construct histone deacetylase1(HDAC1) plasimid (pcDNA3.1-hdac1) by subcloning technology;(our previous work has been completed)1.2To make competence bacterium (E.coli) by CaCl2method;1.3To transform pcDNA3.1-hdacl plasimid into competence bacterium (E.coli), harvest large number of pcDNA3.1-hdacl plasimid, purify and check it by the enzyme digestion;1.4To transfect the TNBC cell with pcDNA3.1-hdacl plasimid, detect the protein expression level with the western bloting;1.5To examine the change of TNBC cell cycle and the expression of four protein (cylin A2, cylin D2, P21,14-3-3sigma) after transfection of pcDNA3.1-hdacl plasimid by using Flow cytometry (FCM).1.6To examine the change of the four protein expression cylin A2, cylin D2, P21, and14-3-3sigma by using the western bloting.2. Effect of histone deacetylase inhibitor on triple negative breast cancer cell proliferation (Negative intervention)2.1To observe the change of TNBC cell proliferation after being treated by histone deacetylase inhibitor SAHA under inverted microscope.2.2To calculate the change of TNBC cell cycle after being treated histone deacetylase inhibitor SAHA by MTS method.2.3To examine the change of TNBC cell cycle and four protein after treatment of histone deacetylase inhibitor SAHA by using Flow cytometry (FCM).2.4To examine the change of four protein after treatment of histone deacetylase inhibitor SAHA by using western blotting. 【Results】1. Effect of histone deacetylase1(HDAC1) on triple negative breast cancer cell proliferation (Positive intervention)1.1Construction and identification of histone deacetylase1(HDAC1) plasimid (pcDNA3.1-hdacl):After successfully constructing of histone deacetylase1(HDAC1) plasimid (pcDNA3.1-hdac1), we confirmed that hdacl DNA fragment is1449bp (cutting position spot is BamH I/EcoR I)by the enzyme digestion; Western bloting showed HDAC1protein expression in TNBC cell lines after transfection of HDAC1plasimid.1.2Effect of histone deacetylase1(HDAC1) on TNBC MDA-MB-468cell cycle: before transfection of HDAC1plasimid into cells: Flow cytometry(FCM)showed: G1phase cell proportion (60.2±0.3)%, S-phase cell proportion (13.7±0.2)%.after transfection of HDAC1plasimid into cells, Flow cytometry(FCM)showed: G1phase cell proportion dropped(45.7±0.2)%, S-phase cell proportion increased (36.1±0.3)%,; Compared between the two cell lines, P<0.05.1.3Study of the influence of histone deacetylase1(HDAC1) on four proteins. FCM showed:after transfection of HDAC1plasimid into TNBC cells, the protein CyclinA2increased over20%; the protein CyclinD2、P21、14-3-3σ decreased; compared between transfected and control group, P<0.05. The western blotting also showed the same result.2. Effect of histone deacetylase inhibitor (HDACi) on TNBC cell proliferation.(Negative intervention)2.1MTS showed after24h treatment of SAHA, the cell demonstrated growth inhibition and cell apoptosis, the greatest inhibition happened at72h after treatment; FCM showed after adding histone deacetylase inhibitor SAHA on MDA-MB-468cell48h, S-phase cell proportion decreased, the expression of CyclinA2decreased, the expression of Cyclin Dl、P21、14-3-3σ increased, also the cell apoptosis increased. Compared to control group, P<0.05. The western blotting also showed the same result. 【Conclusions】1. The histone deacetylase1(HDAC1) gene express in TNBC cell line, its expression can promote cancer cell growth;2. SAHA is an efficient inhibitor for the proliferation of TNBC cells and can promote their cell apoptosis. Its inhibition is related on time. Tumor suppressor gene P21、14-3-3σ and Cyclin A2、cyclinDl participated in the regulation of SAHA on the cell proliferation.更多还原