【作者】 操胜；

【导师】 杨增岐； 孟颂东；

【作者基本信息】 西北农林科技大学 ， 预防兽医学， 2010， 博士

【摘要】 乳腺癌为女性最常见的恶性肿瘤之一,全世界每年约有120万人新患乳腺癌,约50万人死于乳腺癌。近年来,在我国城市地区的乳腺癌发病率和死亡率也呈明显上升趋势。乳腺癌是一种全身性疾病,术后复发转移率高,即使处于癌症早期,也有潜在转移的可能性。当乳腺癌发生转移时,目前无特效治疗方法,以死亡为转归。因此,探讨肿瘤发病机制,寻找新的治疗靶点,及可用于诊断和预后判断的生物标志物,是当今肿瘤学研究的重要内容。HER2/neu是一种重要的原癌基因,其表达产物属于EGF受体家庭2号成员。该家族共有4名成员,分别为HER1、HER2、HER3和HER4,各成员具有相似的分子结构和功能。EGF受体家族成员通过配体形成同源或异源二聚体,活化胞内酪氨酸激酶,从而激活信号转导通路。EGFR家族介导的胞内信号途径主要有4条,调控细胞的增殖、分化、迁移、粘附、存活及凋亡等uPAR为尿激酶纤溶酶原受体,是一种高度糖基化膜蛋白,由糖基化磷脂酰肌醇( GPI )锚定在细胞膜表面。uPAR在多种细胞和组织中表达,包括肿瘤组织及其细胞系。在肿瘤细胞中,uPAR的过表达预示肿瘤的恶性程度高和不良预后。uPAR是一个多功能的细胞表面分子,不但,能在细胞表面富集uPA,激活纤溶酶原系统,引起细胞外基质(ECM)蛋白水解,促进细胞转移与侵袭,而且,还能与细胞表面其它分子,如整合素和甲酰甲硫氨酸受体及玻璃粘连蛋白等相互作用,调控细胞粘附、增殖、凋亡、以及肿瘤休眠过等过程。Herceptin是一种针对HER2的人源化单克隆抗体,能够特异性识别HER2蛋白的胞外功能区,抑制其介导的信号传导通路。Herceptin是美国FDA批准的第一种分子靶向药物,为乳腺癌尤其是HER2过表达乳腺癌的临床治疗带来了新的突破。临床研究证实,Herceptin主要应用于HER2基因过表达的乳腺癌患者群。Herceptin单独使用对HER2基因扩增的患者有效率约25%,与化疗药物联用有效率为50%左右。患者使用Herceptin治疗后,平均能够延长1年左右的生存期。但是,对HER2基因低表达乳腺癌患者群,疗效较差。先前的研究显示,HER2和uPAR在表达上存在相互影响作用。所以,本研究利用RNA干扰技术初步探讨HER2与uPAR在表达上是否存在相互调控关系;并在体外和体内水平进一步研究了以HER2和uPAR为靶标,对乳腺癌协同治疗。(1)以人HER2和uPAR基因为靶基因,设计和构建了分别单独针对HER2与uPAR基因的RNAi表达载体(pHER2和puPAR)和同时针对HER2和uPAR基因的RNAi表达载体(pHu),将其转染乳腺癌细胞,并用real time PCR和western blotting检测其抑制靶基因表达的效果。结果显示,我们成功构建的RNAi表达载体,并证实其在乳腺癌细胞中能高效抑制靶基因的表达。(2)将重组RNAi表达载体转染乳腺癌细胞,用real time PCR、western blotting和IF同时检测每个转染组中两种靶基因的表达情况,从而,探讨HER2与uPAR在表达上的相互关系。结果显示,将重组质粒pHER2转染细胞时,不但能高效抑制HER2表达,同时能够部分的抑制uPAR的表达;同样,将puPAR转染细胞时,不但能高效抑制uPAR表达,同时能够部分的抑制HER2的表达;将pHu转染乳腺癌细胞,与pHER2和puPAR相比,对HER2或uPAR基因的表达抑制作用更为显著。(3)利用RNA干扰技术单独或同时抑制HER2和uPAR基因的表达,并检测其对乳腺癌细胞增殖、凋亡、周期、侵袭力以及MAPK信号调控通路活化的影响。结果显示,单独或同时敲降HER2和uPAR基因在乳腺癌细胞中的表达时,都能抑制细胞的增殖、促进细胞的凋亡、降低细胞的侵袭力、抑制MAPK信号通路的活化以及使乳腺癌细胞G0/G1发生阻滞。同时敲降HER2和uPAR基因表达与单独敲降HER2或uPAR基因表达相比,其效果更为显著。(4)将Herceptin和puPAR单独或联合用药,在细胞水平探讨其对乳腺癌细胞增殖、凋亡、周期、侵袭力以及MAPK信号调控通路活化的影响。结果显示,将Herceptin和puPAR单独或联合处理乳腺癌细胞时,都能抑制细胞的增殖、促进细胞的凋亡、降低细胞的侵袭力、抑制MAPK信号通路的活化以及使乳腺癌细胞G0/G1发生阻滞。联合用药与单独用药相比,其效果更为显著。(5)将Herceptin和puPAR单独或联合用药,在体内水平探讨其对荷瘤鼠的治疗作用。动物试验表明,Herceptin对HER2低表达、uPAR高表达型肿瘤治疗作用不明显,puPAR对肿瘤的生长有很好的抑制作用。当Herceptin与puPAR联合用药时,对荷瘤鼠有更为显著的治疗作用。综上所述,本研究初步证实HER2和uPAR在表达上确实存在调控关系。同时敲降HER2和uPAR基因表达与单独敲降HER2与uPAR表达相比,对抑制细胞的增殖、促进细胞的凋亡和降低细胞的侵袭力等具有叠加或协同作用。体外和体内试验证实,敲降uPAR基因能够提高乳腺癌细胞对Herceptin治疗的敏感性。更多还原

【Abstract】 Breast cancer is one of the most frequent malignant tumors in female. There are about 120 million women suffering from breast cancer, about 50 million women die of this disease all of the world each year. In recent years, the morbidity of breast cancer was increased sharply in the cities of China. Breast cancer is a systemic disease, the recurrence and metastasis rates are very high after surgical operation, and even the primary tumors have the possibility of metastasis. There are no effective therapy methods for breast cancer after the tumor metastasis to a distant organ. Therefore, to study the pathogenesis of cancer, explore the diagnostic and prognostic biomarkers, and find new therapeutic targets is an important oncology research content in today.HER2 is an important oncogene, its expression product belongs to the epidermal growth factor receptor (EGFR) family. The EGFR family includes four members of HER1, HER2, HER3 and HER4. All of the members have similar moecule structures and functions. EGFR family members binding with ligands to form homo-dimers or hetero-dimers activate intracellular tyrosine kinase, which further activate cysto-signal transduction pathway. EGFR family mediate four mainly cysto-signal transduction pathways, which regulate diverse biological responses, such as proliferation, differentiation, cell motility, and survival.Urokinase-type plasminogen activator receptor (uPAR) is highly glycosylated, extracellular protein, be attached to the cell surface by a GPI anchor. CD87 is a broadly expressed protein, including expression in tumor tissue and cell lines. In tumor cells, uPAR over-expression indicates the worsening of malignant tumors and poor prognosis. uPAR is a multifunctional surface molecular and has the principle function of retaining and concentrating uPA at the cell surface for conversion of plasminogen to plasmin, which exerts its primary role of pericellular proteolysis by activating several metalloproteinases. This process is critical for chemotaxis and cell migration. uPAR can interact with some cell surface factors (integrin formyl Methionine receptor and Vitronectin) and play an important role in cell adhesion, proliferation, apoptosis, and tumor dormancy.Herceptin is a recombinant humanized monoclonal antibody against the HER2 protein that can specifically recognize and bind to the extracellular domain of HER2 molecule. The mechanisms of the action of this antibody involve inhibition of signal transduction. As the first molecular drug, which was approved by FDA, Herceptin bring a new break-through for breast cancer clinical treatment, especially for HER2/neu positive breast cancer patients. Clinical study confirmed that Herceptin mainly was used to treat HER2 over-expression breast cancer patients. When Herceptin was used to cure HER2 gene amplification patients alone, the efficiency is about 25%; when Herceptin combined with chemotherapy, the efficiency is about 50%. After treatment of Herceptin, the average survival period of patients can be extended one year. However, for HER2 low expression breast cancer group, Herceptin has a poor treatment outcome.Previous studies have shown HER2 and uPAR expression exist mutually influence. So, we used RNAi technology to study if HER2 and uPAR could regulate protein expresson mutually; and explore synergistic therapy for breast cancer targeting to HER2 and uPAR.(1) With human HER2 and uPAR gene as a target gene, design and construct the specificity RNA interference plasmid vectors pHER2, puPAR and pHu. pHER2 and puPAR target to HER2 and uPAR respectively; Among them, pHu target both HER2 and uPAR. Then, use real time PCR and western blotting to detect the effect of inhibition target genes expression. The results displayed that we successfully constructed four RNAi plasmids. pHER2 and puPAR can inhibite HER2 or uPAR gene expression respectively in breast cancer cells; and pHu can inhibit both HER2 and uPAR genes expression. Compare pHu with pHER2 or puPAR, pHu has a high efficiency of inhibition HER2 or uPAR gene expression in breast cancer cells.(2) Transfect pHER2, puPAR and pHu to breast cancer cells, and use real time PCR, western blotting, and IF to detect HER2 and uPAR genes expression levels in each transfection group. The results showed that transfected pHER2 to breast cancer cells not only strongly inhibited HER2 expression, but also partly inhibited uPAR expression. Likewise, when puPAR was transfected to breast cancer cells, not only strongly inhibited uPAR expression, but also partly inhibited HER2 expression. Compare pHu with pHER2 or puPAR, pHu has prominence efficiency in inhibition both HER2 and uPAR genes expression.(3) pHER2, puPAR and pHu was transfected to breast cancer cells, then detected and analyzed breast cancer cells proliferation, apoptosis, cycles, invasiveness, and regulation of MAPK signaling pathway activation. The results displayed that pHER2, puPAR and pHu was transfected to breast cancer cells respectively, which all could inhibit cells proliferation, promote apoptosis, reduce cell invasiveness, inhibit activation of MAPK signaling pathway and make cells G0/G1 arrest. But pHu has a stronger efficiency than pHER2 or puPAR.(4) Breast cancer cells were treated with Herceptin, puPAR or both Herceptin and puPAR respectively. Then, breast cancer cells proliferation, apoptosis, cycles, invasiveness, and regulation of MAPK signaling pathway activation were detected and analyzed. The results showed that breast cancer cells were treated with Herceptin, puPAR or both Herceptin and puPAR respectively, which all could inhibit cells proliferation, promote apoptosis, reduce cell invasiveness, inhibit activation of MAPK signaling pathway and make cells G0/G1 arrest. But when breast cancer cells were treated with both Herceptin and puPAR has a stronger efficiency than Herceptin or puPAR alone.(5) Tumor-bearing mice were treated with Herceptin, puPAR or both Herceptin and puPAR, respectively. Then the Anti-tumor effect was detected and analyzed. The results of animal experiments showed that for HER2 low expression breast cancer cell line, puPAR has a strong effect to inhibit tumor growth, but Herceptin just has a low effect. When tumor-bearing mice were treated with both Herceptin and puPAR have a more significant addition or synergistic therapeutic effects than tumor-bearing mice were treated with Herceptin or puPAR alone.In conclusion: HER2 could regulate uPAR expression and uPAR also could regulate HER2 gene expression; Simultaneity inhibition HER2 and uPAR expression has a more significant addition or synergistic effects than inhibition HER2 or uPAR alone on inhibition cells proliferation, promotion apoptosis, decrease cell invasiveness, inhibition activation of MAPK signaling pathway and making cells G0/G1 arrest; Knocking down uPAR gene can increase the sensitivity of breast cancer cells to Herceptin.更多还原