【作者】 魏杨辉；

【导师】 吴爱国；

【作者基本信息】 南方医科大学 ， 普通外科学， 2013， 博士

【摘要】 研究背景和目的乳腺癌是女性最常见的恶性肿瘤之一。在我国占全身各种恶性肿瘤的7%-10%。近年来在我国,特别是在一些大城市及沿海经济发展较快的地区,发病率呈迅速上升趋势,是危害妇女健康的主要疾病。回首过去的二十年,乳腺癌的诊治取得了长足的进展,尤其是在外科治疗方面的发展取得了较好的效果。与此同时,对乳腺癌生物学行为的认识也不断深入,利用现代分子生物学技术对肿瘤的发生、发展进行了进一步的阐述,针对乳腺癌的预后,尤其是已发生侵袭转移的晚期患者预后较差,侵袭转移机制亦已成为当今的研究热点。众所周知,恶性肿瘤的侵袭转移是个多因素、多步骤的进展过程,但其具体机制仍不清楚。全世界每年约有120万妇女患上乳腺癌,占所有女性肿瘤的18%,其中超过17万的患者是三阴性乳腺癌(triple-negative breast cancer, TNBC),占乳腺癌患者的10%-23.8%。TNBC具有侵袭性强、复发早、进展快、生存时间短等特殊临床生物学行为,且对大多数内分泌治疗及乳腺癌的靶向治疗(针对HER-2过表达和雌孕激素受体表达阳性)不敏感,预后较其他类型乳腺癌差。传统的肿瘤治疗模式(手术、放疗、化疗)依然是目前肿瘤治疗的主要手段。然而,随着对肿瘤细胞生长、增殖、凋亡的分子机制研究的深人,伴随着分子生物学革命性的进展,肿瘤的治疗已进人生物治疗时代,使人类靶向性治疗肿瘤成为可能。靶向治疗用肿瘤细胞可以表达,而正常细胞很少或不表达的特定基因或基因的表达产物,形成相对或绝对靶向,最大限度地杀伤肿瘤细胞,而对正常细胞损伤很小的治疗方法。乳腺癌分子靶向治疗是指针对乳腺癌发生、发展有关的癌基因及其相关表达产物进行治疗。用于治疗乳腺癌的分子靶向药物通过阻断肿瘤细胞或相关细胞的信号转导,来控制细胞基因表达的改变,而产生抑制或杀死肿瘤细胞。由于三阴乳腺癌组织学分级较差,不表达ER,PR和HER-2,且多伴有p53突变、EGFR的表达,目前较常用的分子靶向药物不针对这个亚型,尽管该亚型对新辅助化疗有较高的总反应率和病理缓解率,但总体预后仍为最差。目前尚无TNBC的治疗指南,其治疗一般按常规治疗标准进行。近年来,许多研究者启动了多个国际、多中心临床研究,认为三阴乳腺癌主要选择紫杉类、铂类、蒽环类及分子靶向治疗药物。目前寻找TNBC的新治疗靶点是当今世界研究的难点和热点。近年有多项研究表明抑制Galectin-3表达可以影响肿瘤细胞的增殖凋亡,鲜有针对乳腺癌尤其是三阴性乳腺癌的研究。Galectin-3是一种半乳糖苷结合蛋白,属于凝集素家族,它在细胞与细胞间及细胞基质相互作用、细胞生长、细胞周期调控、细胞凋亡、细胞损伤和修复过程,肿瘤转化、转移等过程中扮演了一个重要角色。近年来,几项研究证明了Galectin-3在人类肿瘤中过度表达,包括大细胞淋巴瘤、结直肠癌、乳腺癌、肝癌、脑部肿瘤、黑色素瘤、甲状腺癌。Survivin是一个调亡抑制基因,具有调节细胞增殖和调亡的能力,位于人类染色体17q25,有4个外显子和3个内含子,编码全长1417kb。研究发现Survivin表达在所有常见的人类肿瘤内,从35%的胃癌患者到几乎100%的恶性黑色素瘤患者中均可检测到。在大多数病例中,Survivin的表达预示着较差的预后。,Survivin主要表达于胚胎、发育的胎儿组织和大多数肿瘤组织内,而不表达于成熟的正常组织。Ki-67表达在细胞周期的所有阶段除了Go,它可以用来评估生物行为和增殖活性的细胞。这种过度表达的Ki-67已经被观察到在许多恶性肿瘤。自从1980年代早期被发现以来,Ki-67一直是肿瘤增殖标志物的研究热点,尤其在淋巴瘤、乳腺癌、内分泌和脑癌。它常作为一个补充的分级系统,包括提示增殖信号的有丝分裂计数。Ki-67是Oncotype评分中的五个扩散基因之一,此外还有16个癌症相关基因,共同检测肿瘤复发。本课题首先从半乳糖凝集素-3(Galectin-3)、生存蛋白(Survivin)、Ki-67的角度研究乳腺癌侵袭转移的机制,以指导临床上的诊断、分类、预后和治疗。本实验接着从RNA干扰的角度探讨经过干扰后Galectin-3与三阴性乳腺癌增殖和凋亡的关系,了解Galectin-3在三阴性乳腺癌中的生物学作用,为其能否作为三阴性乳腺癌的基因诊断和治疗的靶点提供依据。此外,乳腺癌5年生存率约50～60%,近50%患者治疗后复发转移,而晚期乳腺癌患者平均生存时间仅18～30个月。治疗乳腺癌虽然有多种方案可选择,然而预后均不理想,副作用大,且乳腺癌易乳腺外转移,因而成为治疗上最困难的肿瘤之一。传统的治疗方法仅能杀死肿瘤细胞中处于分裂期的细胞,一旦治疗停止,肿瘤干细胞仍会继续增殖和分化,导致肿瘤的复发或转移,这就是“蒲公英”现象。2003年,Al-Hajj等在乳腺癌患者的原发病灶中进行了分子标记,发现具有CD44+/CD24-/Lineage-表型的乳腺癌细胞在免疫缺陷动物模型中具有显著的致瘤能力,其移植瘤的细胞组成与原发灶肿瘤相同,在经过多代细胞培养之后,还具有较强的致瘤性,提示这类细胞具有与乳腺干细胞类似的自我更新和分化的本质,将其称为乳腺癌干细胞。乳腺癌干细胞(breast cancer stem cells, BCSCs), BCSCs被认为是肿瘤发生、发展与维持的基础,与肿瘤的复发、转移和对治疗的耐受有关。目前研究认为只有少数BCSCs进入细胞周期,而多数BCSCs处于静止状态,故BCSCsRg常规的化疗药物并不敏感。现有的物理、化学药物治疗手段所针对的是大多数的肿瘤细胞,对肿瘤中少数的BCSCs无法达到最有效的杀灭效果,不能防止恶性肿瘤的复发及转移,而肿瘤的复发与转移是患者死亡的主要原因。现有的乳腺癌治疗方法多旨在尽可能杀死或清除一切肿瘤细胞,但是对存在于肿瘤中的数量稀少的干细胞样细胞群却作用甚微,治疗后残存肿瘤干细胞的增殖,足以促使肿瘤复发和转移。乳腺癌干细胞高表达表面标志物ABC蛋白、MDR. ABCG2等,但是目前尚未找到特异性的靶向治疗标志物。目前,乳腺癌干细胞的靶向治疗研究主要有：(1)DNA损伤修复阻断剂,如ADP核糖聚合酶1(PARPI)阻断剂；(2)细胞表面受体,如EGFR、C-Kit;(3)血管表皮生长因子受体vascular endothelial growth factor(VEGF) receptor抑制剂；(4)Src激酶抑制剂；(5)哺乳类动物雷帕霉素靶蛋白mammalian target of rapamycin(roTOR);(6)白细胞介素IL；(7)肿瘤坏死因子相关凋亡诱导配体tumor necrosis factor-related apoptosis inducing ligand(TRAIL)等。人类黑色素瘤分化相关基因-7/白介素-24(Melanoma diffrentiation-associated gene-7/interleukin-24, MDA-7/IL-24)是一个新型的、具有细胞因子样特性的肿瘤抑制基因,是IL-10细胞因子家族的新成员。它编码分泌型蛋白产物(secreted MDA-7/IL-24protein, sMDA-7/IL-24),与免疫系统的白介素功能相同,并具有抑制肿瘤生长及特异性诱导肿瘤细胞凋亡的能力。目前的实验研究表明IL-24在抑制和分化肿瘤细胞,促进肿瘤细胞凋亡；同时抑制肿瘤周围血管形成；增强肿瘤细胞对放疗、化疗的敏感性；抑制肿瘤细胞的侵袭和转移等方面具有重要作用。IL-24具有多效性、选择性、重叠性、协同性、拮抗性和网络性双重性作用特点。但在研究中发现IL-24并不能完全消灭种植在裸鼠身上的肿瘤。解决这一问题的方法有二种：(1)与其他细胞因子联合；(2)使细胞因子可以稳定高效地表达作用在肿瘤部位。有报道指IL-24能诱导并增强肿瘤坏死因子相关的凋亡诱导配体(Tumor Necrosis Factor-related related apoptosis inducing ligand, TRAIL)的活性。肿瘤坏死因子相关凋亡诱导配体(Tumor necrosis factor-Related Apoptosis Inducing Ligand, TRAIL)或称凋亡素2配体(Apo-2ligand, Apo2L)是由Wiley等从人心肌cDNA文库中克隆出来的,因其氨基酸顺序具有TNF超家族的结构特征并能诱导Jurkat细胞和EB病毒转化的人类淋巴细胞凋亡而得名,因其能诱导不同来源的肿瘤细胞以及病毒转化的细胞发生凋亡,而对正常组织细胞不具有毒性,不会导致正常细胞发生凋亡,同时与其它抗肿瘤药物联合应用能协同杀伤肿瘤细胞,所以成为肿瘤治疗领域的研究热点,是一种新型的抗肿瘤药物。目前的研究认为TRAIL主要通过位于细胞膜的死亡受体DR4和DR5通过其胞外区与TRAIL结合后活化,其胞内死亡功能域DD传导凋亡信号,活化了细胞内的FADD-caspase途径,激活线粒体非依赖性和线粒体依赖性二种途径介导凋亡的发生。本课题最后拟重组构建IL-24-TRAIL的基因载体,尝试解决细胞因子在治疗乳腺癌干细胞中存在的疗效差、副作用大以及安全性不保障等方面的缺点,为乳腺癌干细胞的基因治疗提供新的实验和理论依据,使广大的乳腺癌患者尤其三阴耐药乳腺癌因本项目而受益。本项研究具有潜在的巨大社会效益和经济效益。方法1. Galectin-3在乳腺癌组织中的表达及与Survivin、Ki-67的相关性1.1采用免疫组化SP法检测120例乳腺癌组织,120例癌旁乳腺正常组织中Galectin-3、Survivin、Ki-67的表达。1.2比较Galectin-3、Survivin和Ki-67表达水平与乳腺癌临床病理特征的关系。1.3Spearman秩检验分析乳腺癌中Galectin-3和Survivin表达的相关性。1.4Spearman秩检验分析乳腺癌中Galectin-3和Ki-67表达的相关性。1.5Spearman秩检验分析乳腺癌中Survivin和Ki-67表达的相关性。2.RNA干扰技术抑制Galectin-3表达对三阴性乳腺癌细胞MDA-MB-231增殖、凋亡和侵袭的影响2.1设计并化学合成Galectin-3小干扰片段并瞬时转染乳腺癌细胞系MDA-MB-231(为Galectin-3siRNA组)。2.2以未转染细胞为空白对照,以转染无关siRNA为阴性对照,荧光倒置显微镜观察感染效率,实时定量PCR检测干扰结果。2.3XTT方法观察干扰后24,48,72和96h各组细胞增殖吸光度(A值),流式细胞仪检测各组细胞凋亡情况。2.4划痕实验和Transwell实验比较不同组间细胞迁移和侵袭情况。3. IL-24-TRAIL基因载体的构建及其对乳腺癌干细胞迁移和凋亡的影响3.1以人胎盘组织总RNA为模板,采用RT-PCR二步法,扩增TRAIL的cDNA序列,将其克隆入pIRES载体。3.2扩增IL-24的cDNA序列,将其克隆入pIRES-TRAIL载体,构建其真核瞬时表达载体pIRES-IL-24-TRAIL。3.3以Lipofectamine2000转染技术,将TRAIL、IL-24基因导入乳腺癌干细胞MCF-7和MDA-MB-231。3.4流式细胞仪检测两组细胞凋亡情况。3.5划痕实验比较不同组间细胞迁移情况。4.统计分析均采用SPSS13.0统计学软件包分析和处理实验结果数据,采用Chi-square test分析Galectin-3、Survivin、Ki-67蛋白表达与临床病理资料(包括病人年龄、性别、肿瘤分级及TNM分期)之间的联系。用Pearson列联系数(C)和配对资料的卡方检验,进一步分析Galectin-3、Survivin、Ki-67蛋白三者的相关性。细胞实验数据均以x±s表示,多组间比较采用单因素方差分析(one-way ANOVA):进行方差齐性检验,各组间反应变量为方差齐性,则组间比较采用LSD法,若方差不齐采用近似方差分析Brown-Forsythe法进行校正,采用析因分析明确分组与时间点之间是否存在交互作用,以P<0.05确定为差异具有显著性,双侧检验。结果1. Galectin-3在乳腺癌组织中的表达及与Survivin、Ki-67的相关性1.1120例乳腺癌组织中Galectin-3和Ki-67在乳腺癌组织中表达高于癌旁正常组织(75.00%VS8.33%,χ2=109.70,P＜0.001；62.50%VS6.67%,χ2=82.68,P<0.001),并与乳腺癌病理分级、腋窝淋巴结转移有关(χ2=8.571,P=0.014；χ2=4.444,P=0.035；χ2=6.834,P=0.033；χ2=18.809,P<0.001),差异具有统计学意义,但与年龄、肿瘤大小无显著相关(χ2=0.012,P=0.913；χ2=0.212,P=0.645；χ2=0.192,P=0.661；χ2=0.042,P=0.837)。1.2Survivin蛋白的阳性表达率为70.83%,显著高于癌旁正常组织5.00%(χ2=110.50,P＜0.001),且与淋巴结转移有关(χ2=9.076,P＜0.001),差异具有统计学意义,但与年龄、肿瘤大小,肿瘤组织分级均无显著相关性(χ2=0.003,P=0.959；χ2=0.156,P=0.925；χ2=0.048,P=0.827)。1.3乳腺癌组织中Galectin-3、Survivin和Ki-67三者间两两呈正相关(χ2=24.27,C=0.41,P＜0.001；χ2=27.23,C=0.43,P＜0.001；χ2=26.18,C=0.42,P＜0.001)。2.RNA干扰技术抑制Galectin-3表达对三阴性乳腺癌细胞MDA-MB-231增殖、凋亡和侵袭的影响2.1转染后24h,荧光倒置显微镜显示转染效率为80.60±5.62%,实时定量PCR提示Galectin-3siRNA转染后相应Galectin-3的表达显著下降(0.077±0.003VS0.072±0.004VS0.016±0.001,F=479.7,P<0.001)。2.2干扰后24,48,72和96h空白组和阴性对照组细胞继续生长,Galectin-3siRNA组细胞生长抑制,数量逐渐减少(1.61±0.05、1.86±0.04、2.04±0.26、2.80±0.07VS1.60±0.07、1.77±0.10、2.01±0.37、2.49±0.15VS1.18±0.04、0.97±0.02、0.68±0.15、0.46±0.12),干扰时间和实验分组之间存在交互效应(F=39.18,P＜0.001)。2.3流式细胞术分析发现,Galectin-3siRNA组比空白对照组、阴性对照组凋亡率高(36.99%±0.27%VS2.22%±0.03%VS3.84%±0.05%),差异有统计学意义(F=44058.82,P＜0.001)。2.4Transwell侵袭实验显示转染Galectin-3siRNA的乳腺癌细胞浸润穿过Matrigel膜的细胞数比空白对照组和阴性对照组的细胞数(154.70±24.01VS465.00±31.76VS322.30±23.18),差异有统计学意义(F=102.30,P<0.001)。细胞划痕实验显示Galectin-3siRNA组细胞迁移能力显著低于空白对照组和阴性对照组(愈合率：48.39%±1.63%VS92.62%±2.88%VS88.49±2.25%,F=308.80,P<0.001)。3. IL-24-TRAIL基因载体的构建及其对乳腺癌干细胞迁移和凋亡的影响3.1克隆到TRAIL、IL-24基因的cDNA序列,成功构建其真核表达载体。3.2流式细胞仪发现,MDA-MB-231细胞组结果显示转染组比空白对照组、阴性对照组凋亡坏死率高,差异有统计学意义(27.91%±1.05%,68.81%±5.53%VS10.54%±1.13%,9.52%±0.95%,F=272.90,P<0.001)。MCF-7细胞组结果显示转染组比空白对照组、阴性对照组凋亡坏死率高,差异有统计学意义(13.69%±1.09%,58.71%±3.03%VS3.31%±0.22%,4.32%±0.47%,F=774.60,P<0.001)。3.3分别对MDA-MB-231、MCF-7细胞进行划痕实验,结果发现48h后空白对照组和阴性对照组细胞运动迁移基本上覆盖了大部分划痕区(愈合率：91.77%±2.25%、87.54%±2.93%；93.34%±2.33%、89.54%±2.15%),而转染组划痕区依然显著存在(愈合率：37.47%±1.53%、30.21%±1.29%；39.8%±1.22%、31.85%±1.33%),表明细胞迁移能力下调(F=716.40,P＜0.001；F=848.10,P＜0.001)。结论1. Galectin-3在乳腺癌组织中的表达及与Survivin、Ki-67的相关性Galectin-3、Survivin、Ki-67蛋白相互协同作用,通过抑制细胞凋亡,对乳腺癌发生和发展起重要作用。2.RNA干扰技术抑制Galectin-3表达对三阴性乳腺癌细胞MDA-MB-231增殖、凋亡和侵袭的影响RNA干扰能抑制Galectin-3的表达以及MDA-MB-231细胞的增殖,并诱导细胞凋亡、降低其迁移侵袭能力。3. IL-24-TRAIL基因载体的构建及其对乳腺癌干细胞迁移和凋亡的影响pIRES-IL-24-TRAIL构建成功,并能诱导乳腺癌干细胞凋亡、降低其迁移能力。创新之处1.乳腺癌的发生发展涉及多个基因及各种调控因子的相互作用,是一个复杂的过程,Galectin-3、Survivin及Ki-67与乳腺癌的预后有显著的相关性,可作为乳腺癌的预后指标,有待进一步研究探讨三者具体的作用机制,为乳腺癌靶向治疗提供了新的靶点。2.乳腺癌的侵袭转移是一个多因素、多步骤的进展过程,本项目从临床、分子细胞学两方面研究乳腺癌侵袭转移的机制,用RNA干扰技术、免疫组化、RT-PCR、凋亡分析等技术进行研究,很有深度和广度,目前国内外有许多关于Galectin-3siRNA在呼吸、消化道等肿瘤治疗上的研究,但少有关于三阴性乳腺癌的研究,本课题具有重大应用价值和理论意义。3.虽然国际上已经开始利用重组IL-24或TRAIL治疗某些恶性肿瘤的实验研究,但是尚未有联合在乳腺癌方面运用的报道,本研究项目拟采用pIRES作为IL-24和TRAIL基因的载体,构建pIRES-IL-24-TRAIL,使其在肿瘤内长时间表达具有生物学活性的IL-24和TRAIL,以提高肿瘤治疗效果,而且因为其具有人源化、分子量小、穿透力强、免疫原性弱和易于大规模制备的特点,在发挥生物导弹强大威力的同时达到较理想的安全性。通过文献检索,目前尚无类似报道。此药物能成功上市将会为广大乳腺癌患者带来福音,具有极大的经济及社会效益。更多还原

【Abstract】 Background and objectiveBreast cancer is one of the most common malignant tumor in women. In our country it is accounted for7%～10%of whole body various malignant tumors. During recent years, in our country, especially in some big cities and coastal economic rapid development areas, the incidence is rising rapidly. It becomes the main disease of harming to women health. Since the past two decades, great progress has been made in the diagnosis and treatment of breast cancer, whose development has been achieved good results especially in the surgical treatment. At the same time, the further understanding of the biological behavior of breast cancer has been made, and the occurrence and development of tumor are further described by modern molecular biology techniques. According to the prognosis of breast cancer, especially the patient with advanced invasion and metastasis who has poor prognosis, invasion and metastasis mechanism has also become the researching hot pot today. It is well known that the invasion and metastasis of malignant tumor has a multi-factor, multi-step process, but the specific mechanism remains unclear.About1.2million women developed breast cancer in the world each year, accounting for18%of all female cancers, in which more than170000are triple-negative breast cancer (TNBC), accounting for10%-23.8%of the patients with breast cancer. TNBC has aggressive, early recurrence, progress fast, short survival time, special clinical biological behavior, and unsensitive for most endocrine therapy and targeted therapy of breast cancer (for its Her-2expression and female progesterone receptor positive expression), worse prognosis than other types of breast cancer. Traditional mode of cancer therapy (surgery, radiotherapy, chemotherapy) still is the principal means of cancer treatment. However, as for further research in the molecular mechanism of tumor cell growth, proliferation, and apoptosis, with the revolutionary progress of molecular biology, treatment of tumor has been into the era of human biological treatment, make human targeted treatment of tumor to be realization. Targeted therapy is a relative or absolute targeted therapy, by using certain genes or gene expression product who can express in tumor cells, but not in normal cells to kill tumor cells maximally and damage to the normal cells minimally. Molecular targeted therapy of breast cancer is a pointer to the breast cancer occurrence and development of cancer gene expression and its related product for treatment.The treatment of breast cancer molecular targeted drugs by blocking tumor cells or related cell signal transduction, to control the change of cell gene expression, and inhibit or kill tumor cells. Because TNBC have poor histologic classification of breast cancer, no expression of ER, PR and its HER-2, and associated with p53mutations, the expression of EGFR, at present the common molecular targeted drugs are useless for this subtype, although the subtypes of neoadjuvant chemotherapy with higher total response rate and pathological response rate, overall is still the worst prognosis.There is currently no TNBC treatment guidelines, and its treatment generally carried out according to the conventional treatment standards. In recent years, many researchers have launched multiple international, multicenter clinical study, think that triple negative breast cancer mainly choose Paclitaxel and cisplatin,, anthracycline-based and molecular targeted therapy drugs. Currently looking for TNBC new therapeutic targets is a difficulty and focus of research in the world.In recent years, several studies have shown that inhibition of Galectin-3expression can influence the proliferation and apoptosis of tumor cells, but few studies for breast cancer, especially triple-negative breast cancer. Galectin-3(Gal-3) is a member of the carbohydrate-binding protein family known as lectins. It plays a role in cell-cell and cell-matrix interactions, cell growth, cell-cycle regulation, apoptosis, cell damage and repair processes, neoplastic transformation, and metastasis. In recent years, several studies have documented overexpression of Gal-3in different human tumors, including large-cell lymphoma, colorectal carcinoma, breast carcinoma, hepatocellular carcinoma, brain tumors, melanoma, and thyroid carcinoma.Survivin is an inhibitor-of-apoptosis gene,described as capable of regulating both cell proliferation and apoptotic cell death. Mapped to chromosome17q25, survivin consists of four exons and three introns, encoding1417kb message. Studies of common human cancers have shown survivin protein expression in all cancer types, ranging from approximately35%survivin-positive patients in gastric to approximately100%in malignant melanomas. In the majority of cases, survivin protein has been associated with poorer prognosis. In comparison to fetal and cancer cells, relatively little expression of survivin has been detected in many normal adult tissues. Ki-67is expressed in all phases of the cell cycle except GO, and it can be used to evaluate the biologic behavior and proliferation activity of cells. The overexpression of Ki-67has been observed in many malignant tumors. Since its discovery in the early1980s, there has been interest in the role of Ki-67as a proliferation marker in cancer, particularly lymphomas, breast, endocrine and brain cancers. It is commonly used as a complement to grading systems that include mitotic counting as a sign of proliferation. Ki-67is one of the five genes of proliferation that contributes an importance weight to the Oncotype score, out of16cancer-associated genes. First, this topic study the invasion and metastasis of breast cancer mechanism by Survivin, Galectin-3and Ki-67, to guide the clinical diagnosis, classification, prognosis, and treatment. This experiment explores the relationship between SiRNAGalectin-3and TNBC in proliferation and apoptosis by RNA interference, and understands the role of Galectin-3playing in biology behavior of TNBC, which supports it becoming the new target for gene diagnosis and treatment of TNBC.In addition,5-year survival rate of breast cancer is about50%-60%, in which nearly50%patients relapse after treatment. Average survival time of the patients with advanced breast cancer is only18-30months. Although there are many alternative treatment of breast cancer, poor prognosis, side effects, and easily invading outside the mammary gland make it become one of the most difficult treating tumors. Traditional treatment only can kill tumor cells in splitting phase, once treatment stops, cancer stem cell will continue to proliferate and differentiate, which leads to tumor recurrence or metastasis. This is the "dandelion" phenomenon.In2003, Al-Hajj, etc in the primary lesion of patients with breast cancer molecular markers, found with CD44+/CD24-/Lineage-phenotype of breast cancer cells in the immune deficiency animal model has significant tumorigenic ability, the transplanted tumor cells and primary focal tumor are the same, after multiple generation cell culture, but also has strong tumorigenicity, indicating that this type is similar to mammary gland stem cell self-renewal and differentiation of the essence, it is called breast cancer stem cells. Breast cancer stem cells (breast cancer stem cells, BCSCs), BCSCs is considered is the basis of tumor occurrence, development and maintenance, and tumor recurrence and metastasis, and resistance to therapy. Current research suggests only a handful of BCSCs enter the cell cycle, and most of BCSCs in static state, so the BCSCsRg conventional chemotherapy drugs is not sensitive. Existing physical, chemical and drug treatment for the majority of tumor cells, the tumor in a handful of BCSCs can’t achieve the most effective to kill effect, can prevent malignant tumor recurrence and metastasis, and tumor recurrence and metastasis is the leading cause of death in patients. Existing breast cancer treatments designed to kill or remove all the tumor cells as much as possible, but for the few stem cells exist in the tumors in the cells, but to little effect, after treatment residual cancer stem cell proliferation, enough to make tumor recurrence and metastasis. Breast cancer stem cell surface markers CD44expression, high protein, MDR, ABC ABCG2, etc., but has yet to find specific targeted therapy of markers.At present, breast cancer stem cell targeted therapy research mainly has:(1) the DNA damage repair blockers, such as ADP ribose polymerase1(PARPI) blockers;(2) the cell surface receptors, such as EGFR, C-Kit;(3) vascular epidermal growth factor receptor vascular endothelial growth factor (VEGF) receptor inhibitors;(4) the Src kinase inhibitors;(5) mammals rapamycin target protein in mammalian target of rapamycin (roTOR);(6) interleukins IL;(7), tumor necrosis factor related apoptosis inducing ligand tumor necrosis factor-related apoptosis inducing ligand (TRAIL), etc. IL-24(Melanoma diffrentiation-associated gene-7/interleukin-24, MDA-7/IL-24) is a new, cell factor characteristics of the tumor suppressor genes, is IL-10new members of the family of cytokines. Its code is secreted protein product(secreted MDA-7/IL-24protein, sMDA-7/IL-24), same as interleukin function of the immune system, and can inhibit tumor growth and specific ability to induce tumor cell apoptosis. The present experimental study showed that IL-24in inhibition and differentiation of tumor cells, promote tumor cell apoptosis; Inhibition of tumor angiogenesis around at the same time; Enhance the sensitivity of tumor cells to radiotherapy and chemotherapy; Inhibition of tumor cell invasion and metastasis plays an important role. IL-24has pleiotropic, selective, overlapping, collaborative, antagonistic, and network duality function characteristics. But in the study found that IL-24is not entirely eliminate cancer grown in nude mice. To solve this problem there are two ways of:(1) and other cytokines joint;(2) make cytokines can efficiently express stable role in the tumor site. Reports to IL-24can induce and enhance the Tumor Necrosis Factor related apoptosis inducing ligand (Tumor Necrosis Factor-related related apoptosis inducing ligand, TRAIL) activity. Tumor necrosis factor Related Apoptosis Inducing Ligand (Tumor necrosis factor-Related Apoptosis Inducing Ligand, TRAIL) element2Ligand or Apoptosis (Apo-Ligand2, Apo2L) is a myocardial cDNA library by Wiley, etc from the cloned, because of its amino acid sequence with TNF superfamily structure characteristics and can induce Jurkat cells and EB virus transformation of human lymphocytes Apoptosis and its name, because of their different sources can be induced by Tumor cells and virus into cells Apoptosis, and has no toxicity to the cells of normal tissue, won’t cause normal cells Apoptosis, and coordination with other antitumor drugs combined application can kill Tumor cells, and so become a research hotspot in the field of Tumor treatment, is a new type of antitumor drugs. Current research think TRAIL through the main death receptor DR4and DR5in cell membrane by its extracellular region combined with TRAIL after activation, the function of intracellular death domain DD signal conduction, apoptosis, and activate the intracellular FADD-caspase pathway, activation of independence in mitochondria and mitochondrial dependent two pathways mediated apoptosis.This experiment intends to restructure an IL-24-TRAIL gene vecter, and try to solve the shortcomings of cytokine treatment for breast cancer stem cells such as poor curative effect, great side effect and unguarantee safety and so on, which provides new experimental and theoretical basis for breast cancer stem cell gene therapy. Make breast cancer patients especially triple negative breast cancer benefit from this project. This study has potential huge social benefit and economic benefit.Methods and materials1. The Expression of Galectin-3in Breast Cancer and the correlation of Galectin-3, Survivin and Ki-67.1.1The expression of Galectin-3. Survivin and Ki-67in breast cancer (120cases) and paracacinoma normal tissues(120cases) were evaluated by SP immunohistochemistry.1.2The relationship between the expression level of Galectin-3, Survivin and Ki-67, and the clinical and pathological features of breast cancer.1.3The correlation of Galectin-3and Survivin expression in breast cancer was analysis Spearman rank test.1.4The correlation of Galectin-3and Ki-67expression in breast cancer was analysis Spearman rank test.1.5The correlation of Survivin and Ki-67expression in breast cancer was analysis Spearman rank test.2. Influences of Inhibiting Galectin-3expression by RNA Interference Technique on the Proliferation, Apoptosis and Invasion of Triple-negative Breast Cancer Cells MDA-MB-2312.1Galectin-3small interfering RNA (siRNA) was constructed by chemistry synthesis. Effective siRNA was transfected into breast cancer cell MDA-MB-231(as Galectin-3siRNA group).2.2Non-transfected cells were used as blank control and negative RNA as negative control. Inverted fluorescence microscope and RT-PCR was used to verify the interference result.2.3Cellproliferation (absorbance value) was observed by XTT after interference for24,48,72and96hours. Cell apoptosis was measured with flow cytomter.2.4Wound healing test and transwell test were performed for cell motility and invasion assay.3. Construction of IL-24-TRAIL vector and its influences on the motility and apoptosis of Breast Cancer Stem Cells3.1cDNA fragment encoding human TRAIL gene were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) with the total mRNA isolated from human placenta tissue as template. The PCR amplified fragment of TRAIL gene was cloned into pIRES vector.3.2cDNA fragment encoding human IL-24gene were amplified. The PCR amplified fragment of IL-24gene was subcloned into the recombinant eukaryotic expression vector pIRES-TRAIL to construct pIRES-IL-24-TRAIL plasmid after sequencing.3.3Plasmid DNA of pIRES-IL-24-TRAIL was transfected into Breast Cancer Stem Cells MCF-7and MDA-MB-231with the help of Lipofectamine2000transfection reagent.3.4Cell apoptosis was measured with flow cytometer.3.5Wound healing test was performed for cell motility assay.4. statistical analysisAdopt SPSS13.0statistical software analysis the experimental results. analysis the relationship betewwn Galectin-3, Survivin, Ki-67protein expression and clinical pathological data (including the patient’s age, sex, tumor grade and TNM stage) by the Chi-square test. Using Pearson column connection number (C) and Chi-square test for paired data, analysis further the correlation of Galectin-3, Survivin, Ki-67protein. Experimental data are list as x±s, using one-way ANOVA comparing between groups: homogeneity test for variance first, if the variance is homogeneity, choose LSD method, if the variance is not homogeneity, Brown-Forsythe approximate variance analysis method is used for calibration, using factorial ANOVA analysis whether there is interaction between teams and time points, with P <0.05has significant difference, two tailed test.Results:1.The Expression of Galectin-3in Breast Cancer and the correlation of Galectin-3, Survivin and Ki-67.1.1The expression of Galectin-3and Ki-67in breast cancer was also significantly higher than that in paracacinoma normal tissues (75.00%VS8.33%, χ2=109.70, P<0.001;62.50%VS6.67%, χ2=82.68, P<0.001). The expression of Galectin-3and Ki-67was correlated with the pathological grade and node metastasis (χ2=8.571, P=0.014;χ2=4.444, P=0.035;χ2=6.834, P=0.033;χ2=18.809, P<0.001). There were no correlations among their expressions and the age or tumor size (χ2=0.012, P=0.913;χ2=0.212, P=0.645;χ2=0.192, P=0.661;χ2=0.042, P=0.837)1.2The positive rates of Survivin were70.83%in breast cancer,5.00%in nomal tissues. The positive rate of Survivin expression was significantly higher in breast cancer than that in normal tissues (χ2=110.50, P<0.001), and was correlated with node metastasis (χ1=9.076, P<0.001). There were no correlations between the expression o f Survivin and the age, histological grade or tumor size (χ2=0.012, P=0.913;χ2=0.212, P=0.645;χ2=0.192, P=0.661;χ2=0.042, P=0.837) 1.3The expression of Galectin-3was positively correlated with Survivin and Ki-67(χ2=24.27, C=0.41, P<0.001; χ2=27.23, C=0.43, P<0.001; χ2=26.18, C=0.42, P<0.001)2. Influences of Inhibiting Galectin-3expression by RNA Interference Technique on the Proliferation, Apoptosis and Invasion of Triple-negative Breast Cancer Cells MDA-MB-2312.1After transfected24hours, the transfection efficiency rate by inverted fluorescence microscope was80.60±5.62%. RT-PCR showed that the expression of Galectin-3was markedly decreased in Galectin-3siRNA group after transfected (0.077±0.003VS0.072±0.004VS0.016±0.001, F=479.7, P<0.001)2.2Cells proliferated still in the blank group and negative contro group after24,48,72and96hours, but cells grew inhibited and declined gradually in Galectin-3siRNAgroup(1.61±0.05、1.86±0.04、2.04±0.26、2.80±0.07VS1.60±0.07、1.77±0.10、2.01±0.37、2.49±0.15VS1.18±0.04、0.97±0.02、0.68±0.15、0.46±0.12).There was the interaction effect between the interference time and the experiment group (F=39.18, P<0.001)2.3The apoptosis rate by flow cytomter was slight higher in the Galectin-3siRNA group than the normal and blank controlgroup (36.99%±0.27%VS2.22%±0.03%VS3.84%±0.05%, F=44058.82, P<0.001)2.4The results of Transwell experiment indicated that the number of cells in down-pore of micro-membrane in the transfected group was less than that in the control group(154.70±24.01VS465.00±31.76VS322.30±23.18, F=102.30, P<0.001). Wound-healing assay shows that cell motility could be effectively suppressed by Galectin-3siRNA (Healingrate:48.39%±1.63%VS92.62%±2.88%VS88.49±2.25%, F=308.80, P<0.001) 3. Construction of IL-24-TRAIL vector and its influences on the motility and apoptosis of Breast Cancer Stem Cells3.1cDNA sequence encoding human TRAIL and IL-24gene were successfully cloned, and recombinant eukaryotic expression vector pIRES-IL-24-TRAIL was constructed.3.2The apoptosis rate of MDA-MB-231by flow cytomter was higher in the transfected group than the controlgroup (27.91%±1.05%,68.81%±5.53%VS10.54%±1.13%,9.52%±0.95%, F=272.90, P<0.001). The apoptosis rate of MCF-7by flow cytomter was higher in the transfected group than the controlgroup(13.69%±1.09%,58.71%±3.03%VS3.31%±0.22%,4.32%±0.47%, F=774.60, P<0.001)3.3Wound-healing assay shows that cell motility could be more effectively suppressed in the transfected group than the controlgroup (MDA-MB-231:7.47%±1.53%,30.21%±1.29%VS91.77%±2.25%,87.54%±2.93%, F=716.40, P<0.001; MCF-7:39.8%±1.22%,31.85%±1.33%VS93.34%±2.33%,89.54%±2.15%, F=848.10,P<0.001)Conclusion1. The Expression of Galectin-3in Breast Cancer and the correlation of Galectin-3, Survivin and Ki-67.Galectin-3, Survivin and Ki-67may play an important role in the carcinogenesis and progress of breast cancer through the inhibition of apoptosis. They may play synergetic roles in the process of carcinogenesis of breast cancer.2. Influences of Inhibiting Galectin-3expression by RNA Interference Technique on the Proliferation, Apoptosis and Invasion of Triple-negative Breast Cancer Cells MDA-MB-231 The interference of RNA inhibits the expression of the Galectin-3and the proliferation of MDA-MB-231cells significantly, while it promotes the apoptosis of tumor cells and impede tumour cell invasion.3. Construction of IL-24-TRAIL vector and its influences on the motility and apoptosis of Breast Cancer Stem CellspIRES-IL-24-TRAIL was constructed successfully, and it can promotes the apoptosis of tumor cells and impede tumor cell motility.Innovation1. Breast cancer development involving multiple genes and various regulatory factors interaction, is a complex process. Survivin, Galectin-3and Ki-67have obvious relevance the prognosis of breast cancer, which can be used as prognostic indicators of breast cancer. Further research in the specific inner mechanism of them is needed,which provides a new target therapy for breast cancer.2. The invasion and metastasis of breast cancer is a multi-factor and multi-step process. The project researchs in mechanism of invasion and metastasis in breast cancer by clinical and molecular biology aspects, using RNA interference technique, immunohistochemistry, RT-PCR, apoptosis and analysis technology to research, which is of great depth and breadth. There are many research about the Galectin-3SiRNA in the treatment of respiratory and digestive tract tumor at home and abroad, but there are few studies on triple-negative breast cancer. This reseach has important application value and theoretical significance.3. Although international has started using recombinant IL-24or TRAIL to treat some malignant tumors in experimental studies, but there is no joint used in breast cancer. This research project is proposed pIRES as a carrier of the IL-24and TRAIL gene, building pIRES-IL-24-TRAIL, which can express has activity-biology IL-24and TRAIL in the tumour for a long time, to improve the effect of tumor therapy, and because it is humanized, small molecular weight, strong penetrating power, weak immunogenicity and the characteristics of easy to large-scale preparation, it can achieve the ideal security while exerting biological missile power. Through literature search, there is no similar report. The drug listed successfully will brought good news the patients of breast cancer, which has great economic and social benefits.更多还原