【作者】 刘芳；

【导师】 罗荣城；

【作者基本信息】 南方医科大学 ， 肿瘤学， 2014， 博士

【摘要】 随着分子生物学与免疫学的迅速发展,肿瘤的免疫治疗逐渐成为继手术、化疗和放疗之后的肿瘤治疗新模式。但是,因为实用的特异性肿瘤抗原较少、肿瘤抗原的免疫原性比较低,难以完成抗原的有效递呈,所以,在大多数的肿瘤治疗中,免疫治疗只起到了辅助治疗的作用。因此,寻找特异性的肿瘤抗原并将其作为免疫治疗的潜在靶标成为肿瘤免疫治疗的核心问题。近些年来,胃癌的死亡率虽然在全球呈现一个下降的趋势,但其死亡率仍排在恶性肿瘤的第二位。我国是胃癌的高发区,胃癌的死亡率居于首位。大多数患者诊断时已经处于疾病的晚期,失去手术根治的机会,即使根治性切除术后患者仍有约一半的患者面临肿瘤复发。对于晚期及复发转移的患者,目前常规治疗是放化疗,但是效果并不是很理想。因此,检测肿瘤组织或细胞的特异性分子表达,并以此作为靶点,但同时也不损伤人体正常细胞的肿瘤分子靶向治疗在当今肿瘤治疗领域中的地位越来越重要。肿瘤-睾丸抗原是一类肿瘤抗原,其表达具有高度的组织限制性,一般只表达在正常睾丸和肿瘤组织中。由于生殖细胞不表达人白细胞抗原分子以及血睾屏障的存在,所以肿瘤睾丸抗原在产生抗肿瘤免疫的同时不会对正常组织及生殖细胞产生危害。越来越多的研究发现肿瘤睾丸抗原在各种肿瘤组织中特异性的表达,并且在肿瘤细胞发生、发展、预后等方面发挥重要作用。睾丸特异性蛋白质50是应用甲基敏感差异分析技术从人正常睾丸组织和乳腺癌组织中分离出来的的低甲转移,诱导肿瘤细胞凋亡,甚至可以增强肿瘤细胞对抗肿瘤药物的敏感性。TSP50在胃癌中的表达也有报道,但是其样本量较少,同时关于TSP50与临床病理特征及预后的关系目前未见到报道。方法1.收集手术切除的胃癌病例蜡块334例及对应的癌旁组织蜡块20例,共制成6个组织芯片蜡块,每个蜡块包括一定数量的胃癌组织及癌旁组织。2.免疫组化染检测TSP50蛋白在胃癌组织及癌旁组织中表达。3.单因素Kaplan-Meier分析组织学类型、TNM分期、淋巴结转移、TSP50蛋白表达均与胃癌的预后关系。4.多因素Cox回归模型分析组织学类型、TNM分期、淋巴结转移、TSP50蛋白表达与胃癌预后的关系。5.利用Western blot分析比较TSP50在3例胃癌患者的癌组织及其癌旁组织中的表达差异。结果1.TSP50在胃癌组织、癌旁组织的表达。免疫组化染检测TSP50蛋白在胃癌中表达的中位分值为8,我们设定分分值≥18为高表达,<8为低表达。以此标准,TSP50蛋白在191例胃癌中呈高表达(191/334,57.2%),余下的143例中呈低表达,在20例癌旁组织中均呈低表达。2.单因素Kaplan-Meier分析组织学类型、TNM分期、淋巴结转移、TSP50蛋白表达均与胃癌的预后相关弥漫型胃癌、较晚的TNM分期、有淋巴结转移、高表达TSP50蛋白的胃癌预后明显差于肠型胃癌、较早的TNM分期、无淋巴结转移、低表达TSP50蛋白的病例(P值分别为0.006,0.000,0.003,0.021)。高表达TSP50蛋白病例中位生存年龄为38.02个月,而低表达TSP50组为30.21个月。3.多因素Cox回归模型分析组织学类型、TNM分期、淋巴结转移、TSP50蛋白表达的关系组织学类型、TNM分期、淋巴结转移、TSP50蛋白表达均是胃癌的独立预后因素(组织学类型,P值0.048,95%置信区间为1.078-2.172；TNM分期,P值0.000,95%置信区间为1.348-2.573；淋巴结转移,P值0.001,95%置信区间为0.139-0.590；TSP50蛋白表达,P值0.017,95%置信区间为1.078-2.172)。4. Western blot分析比较TSP50在3例胃癌患者的癌组织及其癌旁组织中的表达差异在3例胃癌患者的癌组织及其癌旁组织,胃癌组织中TSP50表达高于癌旁。结论1.高通量组织芯片是高效、简便和标准化的研究技术,可用于大样本的肿瘤学研究。组织芯片技术稳定,结果可靠,结合临床随访数据可以对患者生存预后进行回顾性分析。2.TSP50蛋白在约57.2%的胃癌组织中呈高表达,且其表达与较年轻的发病年龄、较晚的TNM分期、有淋巴结转移高度相关。3.TSP50蛋白表达可作为胃癌的一项独立预后指标。胃癌是消化系统常见肿瘤,虽然近年来胃癌在发达国家发病率有所下降,但在我国农村情况则仍然严峻,胃癌的死亡率仍然位居恶性肿瘤死亡率的第一位[35],是威胁我国人民健康的主要恶性肿瘤之一。尽管近年来检测方法及治疗手段的得到了长足的发展,但是多年来胃癌治疗后的五年生存率一直低于25%[36,37]。大多数胃癌患者就诊时已达Ⅲ或Ⅳ期,而淋巴结转移率高达50-70%。临床流行病学研究发现,导致胃癌高死亡率的重要原因是胃癌的侵袭转移。因此,寻找和研究胃癌侵袭转移的相关分子和功能性基因,从而获得能用于靶向治疗转移性胃癌及胃癌复发转移的分子靶标的功能性基因。同时也望能获得检测预示胃癌复发转移及预后的分子标志物,为胃癌的临床个体化治疗提供科学依据。研究表明,离子通道在和胞内Ca2+内稳态失衡与肿瘤增殖、凋亡、迁移有密切关系,但其具体机制尚不明晰。钙激活氯通道是由细胞内Ca2+激活的氯离子通道,广泛表达于上皮细胞并参与细胞分泌以及容量性调节,从而维持细胞在增殖、凋亡、迁移等生理过程中的离子平衡。TMEM16A是最近被证实为钙激活氯通道,虽然TMEM16A对细胞内钙离子浓度的依赖性,但是在TMEM16A的基因序列中找不到与钙离子直接结合的结构域或与钙调蛋白结合的结构,那么TMEM16A通道与钙离子的关系是直接结合还是通过某些中间传导通路的介导,目前TMEM16A通道是如何被钙离子激活的分子机制尚未研究清楚。TMEM16A定位于人染色体11q13区域,此区域很多基因在上皮类癌症如乳腺癌、膀胱癌、肺癌、头颈部肿瘤中扩增并表达,并且在肿瘤发生发展过程中均发挥重要作用,这提示我们TMEM16A可能在肿瘤发生发展过程中有一定的作用。同时,Ca2+是细胞增殖、凋亡、及细胞运动不可缺少的因子基础因子,但是其具体机制尚不清楚。TMEM16A是细胞内钙激活的氯离子通道,它极有可能是Ca2+发挥作用的下游分子基础。因此提示TMEM16A在肿瘤的发生发展中可能发挥调节作用,本研究探讨了钙激活氯通道TMEM16A在胃癌发生和发展过程中的作用。本研究首先证实,免疫组化及westernblot实验证实TMEM16A在人胃癌组织、胃癌细胞株中高表达而在人胃癌癌旁组织中低表达或无表达,且其表达与较晚TNM分期、有淋巴结转移高度相关。为了进一步证实TMEM16A与胃癌的关系,我们采用人TMEM16A特异性双链shRNA慢病毒重组载体转染AGS细胞,shRNA被酶切形成siRNA,再与mRNA结合降解后即达到靶基因沉默,抑制AGS细胞内TMEM16A的表达,获得TMEM16A稳定消减的细胞株。最后,通过增殖、划痕以及Transwell小室等实验手段进一步证实,沉默TMEM16A可以明显抑制AGS胃癌细胞迁移和侵袭的能力。方法1.收集手术切除标本制作成组织芯片,共包括367例胃癌病例,其中胃癌病例蜡块367例及对应的癌旁组织蜡块20例,淋巴结转移性胃癌30例。另外制作5例胃癌组织大切片,在同一蜡块上包括癌,癌旁组织及远处正常组织(距癌的距离大于3cm)。2.应用免疫组化检测TMEM16A在胃癌组织、癌旁组织及淋巴结转移性胃癌的表达。3.应用westernblot实验检测TMEM16A在新鲜胃癌组织、癌旁组织。4.采用人TMEM16A特异性双链shRNA慢病毒重组载体转染AGS细胞,shRNA被酶切形成siRNA,再与mRNA结合降解后即达到靶基因沉默,抑制AGS细胞内TMEM16A的表达,获得TMEM16A稳定消减的细胞株。然后,通过增殖、划痕以及Transwell小室等实验手段,沉默TMEM16A后胃癌细胞株增殖、迁移、侵袭能力的变化。5.单因素Kaplan-Meier分析MEM16A蛋白表达与预后的关系；多因素Cox回归模型分析,结果显示肿瘤位位置、组织类型、TNM分期、淋巴结转移、TEME16A蛋白表达与预后的关系。结果1. TMEM16A在胃癌组织、癌旁组织及淋巴结转移性胃癌的表达。通过SPSS19.0分析软件分析训练集(training set)中TMEM16A免疫组化分值,得出ROC曲线坐标表,根据表中数据计算出约登指数,其对应的分值即为免疫组化计分分值最佳临界点。约登指数最大值为0.137,其对应的分值为8.5,即为最佳临界点。根据免疫组化分值的实际值,我们设定大于8为高表达,低于或等于8为低表达。通过在同一组数据分析中,训练集(training set)用来训练模型或确定模型参数,测试集(testing set)则是根据在训练集中所确定的模型参数来进行统计学估算。在训练集中,TMEM16A蛋白在75例胃癌中呈高表达(75/113,66.4%),余下的38例中呈低表达(38/113,33.6%)。在测试集中,TMEM16A蛋白在179例胃癌中呈高表达(179/254,70.4%),余下的57例中呈低表达(75/254,29.6%)。在20例癌旁组织中均呈低表达,在22例淋巴结转移癌中高表达(22/30,73.3%)。在训练集及测试集中,TMEM16A蛋白表达与胃癌的TNM分期、淋巴结转移明显相关,较晚TNM分期及有淋巴结转移的胃癌病例中TMEM16A呈高表达水平(P值分别为：训练集0.007,0.000；测试集0.033,0.001)。5例胃癌组织大切片(同一切片包括胃癌、癌旁及远处正常组织)的免免疫组化也显示由胃癌到远处正常组织的TMEM16A蛋白的染色强度变。2. Westernblot实验检测TMEM16A在新鲜胃癌组织、癌旁组织表达。Westernblot分析显示在12例手术切除的新鲜胃癌标本中,TMEM16A蛋白在胃癌组织中的表达明显高于在癌旁组织的表达。3. TMEM16A与胃癌预后的关系单因素Kaplan-Mete分析分别分析测试集及所有病例中TMEM16A蛋白表达与预后的关系,结果显示高表达TMEM16A蛋白的胃癌病例的预后明显差于低表达的病例(P值分别为：测试集,0.022；所有病例,0.018)。多因素Cox回归模型分别对测试集及所有病例时进行分析,结果显示肿瘤位于全胃、弥漫类型、较晚TNM分期、有淋巴结转移、高TEME16A蛋白表达均分别提示较差的预后,上述因素均可做为胃癌的独立预后因素(肿瘤位置：测试集P值0.019,95%置信区间为1.073-2.193；所有病例,P值0.014,95%置信区间为1.076-1.904。组织学类型：测试集P值0.014,95%置信区间为1.122-2.733；所有病例,P值0.001,95%置信区间为1.303-2.699。TNM分期：测试集P值0.000,95%置信区间为1.581-3.334；所有病例,P值0.000,95%置信区间为1.572-2.933。淋巴结转移：测试集P值0.027,95%置信区间为0.192-0.905；所有病例,P值0.021,95%置信区间为0.246-0.891。TMEM16A蛋白：测试集P值0.026,95%置信区间为0.381-0.942；所有病例,P值0.010,95%置信区间为0.428-0.890)。5.沉默TMEM16A后胃癌细胞株增殖、迁移、侵袭能力的变化用Westernblot检测沉默后的AGS细胞株TMEM16A蛋白的表达,TMEM16A shRNA/AGS组TMEM16A蛋白的表达低于scrambled shRNA/AGS、AGS组,TMEM16A被显著抑制。通过BrdU掺入实验、划痕实验、transwell小室侵袭及迁移实验,提示沉默TMEM16A可降低AGS迁移侵袭能力,但对增殖能力无明显影响。结论1.钙激活氯通道TMEM16A在人胃癌组织中高表达而在胃癌癌旁组织中低表达或无表达,且其表达与较晚TNM分期、有淋巴结转移高度相关。2.抑制钙激活氯通道TMEM16A的表达可抑制AGS胃癌细胞系体外迁移、侵袭能力；3.膜蛋白TMEM16A也许可作为胃癌治疗的潜在靶点和生物标志物。恶性肿瘤最重要的特性就是能够发生侵袭和转移,大部分的恶性肿瘤死亡是肿瘤转移引起的,因此肿瘤侵袭转移也是肿瘤学研究的热点。上皮一间质转化(epithelial mesenchymal transitions, EMT)是指上皮细胞在一定条件下向间质细胞表型转化的过程,使具有极性的上皮细胞转换成具有活动能力、能够在细胞基质问自由移动的间质细胞的过程。EMT是一种基本的生理病理现象,参与胚胎的形成、发育、组织重建和伤口修复,近些年来越来越多的研究表明EMT在肿瘤侵袭和转移中也起着重要用。而E-钙粘素的减少或者丢失是上皮一间质转化过程中最重要的标志性变化。有很多研究表明E-钙粘素的减少或者丢失是胃癌发生侵袭转移的重要因子之一,而在我们前期实验中我们发现TMEM16A与胃癌侵袭和转移密切相关,由此我们推想,TMEM16A与E-钙蛋白表达是否相关呢?方法1.收集手术切除标本制作成组织芯片,共包括367例胃癌病例,其中胃癌病例蜡块367例及对应的癌旁组织蜡块20例,淋巴结转移性胃癌30例。2.免疫组化染检测E-钙粘素在胃癌组织、癌旁组织及转移性淋巴结中表达。3. Phi and Cramers V相关分析在胃癌组织、转移性淋巴结、癌旁组织中E-钙粘粘素与TMEM16A的相关性。4.采用人TMEM16A特异性双链shRNA慢病毒重组载体转染AGS细胞,shRNA被酶切形成siRNA,再与mRNA结合降解后即达到靶基因沉默,抑制AGS细胞内TMEM16A的表达,获得TMEM16A稳定消减的细胞株。然后,比较TMEM16A沉默前后E-钙粘粘素表达情况。结果1.TSP50在胃癌组织、癌旁组织、转移性淋巴结的表达。免疫组化染检测34.1%患者E-钙蛋白在胃癌组织中高表达。2. Phi and Cramers V相关分析在胃癌组织、转移性淋巴结、癌旁组织中E-钙粘粘素与TMEM16A的相关性。Phi and Cramers V相关分析发现在胃癌组织中两者表达呈负相关(r=-0.161,P=0.002),在转移性淋巴结及癌旁病灶两者的表达也是负相关。3.比较TMEM16A沉默前后E-钙粘粘素表达情况。沉默TMEM16A胃癌细胞表达E-钙粘素表达明显下降。结论胃癌组织中TMEM16A与E-钙蛋白表达呈负相关(r=-0.161,P=0.002),在转移性淋巴结及癌旁病灶两者的表达也是负相关。在体外实验,沉默胃癌细胞TMEM16A表达可以使E-钙粘素表达明显下降。本学位论文的第三部分得出如下结论：钙激活氯通道TMEM16A与E-钙粘素表达负相关,其促进侵袭转移的机制可能通过上皮-间质转化完成的。更多还原

【Abstract】 With development of society and economy,morbidity and mortality of malignant tumor increase.Surgery,chemotherapy and radiotherapy are conventional reatments,which can not cure the most cancers,not mention to advanced stage of caners.The effects of conventional treatments are disappointed,and can not meet the needs of patients. Immunotherapy is the most possible to cure the tumors,especially advanced malignant tumor. Nowadays, Immunotherapy is the auxiliary to conventional treatments,because of limitation of specificity and numbers of tumor antigen,then,the most important thing is to find specific tumor antigen.Testes-specific protease50(TSP50) is a testis-specific gene that encodes a protein, which is homologous to serine proteases. Normally, TSP50protein is specifically expressed in the spermatocytes of testes, but abnormally activated and expressed in breast cancer. Currently, TSP50is considered as a member of cancer-testis antigens (CTAs), which include almost140members, such as Melanoma Antigen-Encoding Gene-1(MAGE-1), Cancer/testis antigen cancer-associated gene (CAGE), and Opa Interacting Protein5(OIP5), and so on. These proteins are expressed in various types of human cancers including gastric cancer and may serve as tumor markers for clinical prognosis or targets for therapeutic approaches. In this regard, MAGE-1protein is a predictive marker of poor prognosis in differentiated advanced gastric cancer patients Nakamura et al. revealed that OIP5might be a novel immunotherapy target for patients with gastric cancer. However, the expression of TSP50protein in gastric cancer and its diagnostic and/or prognostic significance has not been elucidated.In this study, the expression of TSP50protein was examined in a large number of human gastric cancer specimens and its clinicopathological and prognositc significance was also assessed.Methods:Immunohistochemistry (IHC) analysis of TSP50was performed on a tissue microarray (TMA) containing334primary gastric cancers and20adjacent non-tumor tissues. Western blot was carried out to confirm the expression of TSP50in3pairs of gastric cancers and adjacent non-tumor tissues.Results:IHC analysis revealed high expression of TSP50in57.2%human gastric cancer samples (191out of334). However, it was poorly expressed in all of the20adjacent non-tumor tissues. This was confirmed by western blot, which showed significantly higher levels of TSP50expression in gastric cancer tissues than adjacent non-tumor tissues. A significant association was found between high levels of TSP50and clinicopathological characteristics including junior age at surgery (P=0.001), later TNM stage (P=0.000), and present lymphnode metastases (P=0.003). The survival rate of gastric cancer patients with high expression of TSP50was significantly shorter than those with low levels of TSP50(P=0.021). Multivariate Cox regression analysis indicated that TSP50overexpression was an independent prognostic factor for gastric cancer patients (P=0.017).Conclusions:Our data demonstrate that elevated TSP50protein expression could be a potential predictor of poor prognosis in gastric cancer patients. Gastric cancer is the second most common cancer in the world, causing nearly one million deaths annually.Although the incidence of gastric cancer has decreased, it still remains among the leading causes of death from cancer in China. Despite major advances in diagnosis and treatment in the past few decades, gastric cancer remains a major clinical challenge.Prognostic factors for survival are useful in the management of gastric cancer. Many molecular markers, such as HER2, E-cadherin, Caveolinl, and so on, have been evaluated as candidate prognostic factors in gastric cancer. However, the prognosis for gastric cancer patients still stays poor and many prognostic factors which can effectively predict the prognosis of gastric cancer patients have not investigated.TMEM16A(transmembrane protein16A), also known as ANO1, DOG1or TAOS2, is an established sensitive biomarker for the diagnosis of gastrointestinal stromal tumors (GISTs). It is a member of the Anoctamin family of membrane proteins, which consists of ten components (known as TMEM16A-K or ANO1-10). Moreover, it has been proved to be a calcium-activated chloride channel, and contribute to many important physiologic functions. The coding sequence of TMEM16A is located within the11q13region, which is one of the most frequently amplified chromosomal regions in human cancers, such as head and neck squamous cell carcinoma, breast cancer and gastric cancer. Furthermore, TMEM16A protein is overexpressed in many tumor types including head and neck squamous cell carcinoma, esophageal cancers, breast cancer and prostate cancer, and it plays a vital role in the tumorigenesis and migration of some cancers. However, the expression of TMEM16A in gastric cancer, its prognostic significance and whether it is required for tumor development and migration has not been elucidated.In the present study, we examined the expression of TMEM16A protein in gastric cancer cell lines and tissues by Western blot analysis, and in gastric cancer tissues microarray(TMA) by immunohistochemistry.The clinical and prognostic significance of TMEM16A expression in gastric cancer was evaluated. Additionally, scratch wound assay and transwell matrix penetration assay are employed to assess the role of knockdown TMEM16A in the proliferation, migration and invasion of gastric cancer cell lines.Methods:Immunohistochemistry (IHC) analysis of TMEM16A was performed on a tissue microarray (TMA) containing367primary gastric cancers and20adjacent non-tumor tissues. Additionally,30matched lymphnode metastatic lesions and5whole-mount section tissues were recruited for this study. Western blot was carried out to confirm the expression of TMEM16A in4pairs of gastric cancers and adjacent non-tumor tissues.Furthermore, scratch wound assay and transwell matrix penetration assay are employed to assess the role of knockdown TMEM16A in the proliferation, migration and invasion of gastric cancer cell lines.Results:IHC analysis revealed strong expression of TMEM16A in66.4%human gastric cancer samples and lymphnode metastasis lesions,while weak expressed in adjacent non-tumor mucosal tissues.Consistent with this result, immunohistochemistry for whole-mount sention and western blot analysis displayed a similar finding in gastric cancer and adjacent non-tumor tissues. By univariate survival analyses using Kaplan-Meier method and log-rank test, the impact of TMEM16A on patient survival was analyzed and we found that elevated expression of TMEM16A was closely associated with poor overall survival in both testing set(P=0.022) and overall patients(P=0.018). Furthermore,scratch wound assay and transwell matrix penetration assay showed that silencing of TMEM16A can inhibit AGS cells to migrate and invade.Conclusions:Our data demonstrate that elevated TSP50protein expression could be a potential predictor of poor prognosis in gastric cancer patients. The most important features of malignant tumors are invasion and metastasis, which contribute to the majority of death because of cancer. Epithelial mesenchymal transitions(EMT) plays vital role in invasion and metastasis of tumors, which is represent of reduction or decrease of E-cadherin. In the previous study we found that TMEM16A is closely relationship with the invasion and metastasis of gastric cancer. To further investigate the underlying mechanism of TMEM16A in the invasion of gastric cell, the relationship between TMEM16A and E-Cadherin, a well-known marker regulating cadherin-based adhesion, was evaluated by immunohistochemistry and Western blot.Then, we speculated that whether or not TMEM16A and E-cadherin are relevant.First, E-Cadherin protein was detected by immunohistochemistry in gastric cancer TMA. High E-Cadherin expression was detected in34.1%(125of367) gastric cancer tissues according to the cutoff point generated by ROC curve analysis. Phi and Cramers V correlation analysis indicated that a significant negative correlation between expression of TMEM16A and E-Cadherin (r=-0.330, P=0.000). Moreover, similar negative relation was investigated in lymph node metastases lesions and adjacent non-tumor mucosa tissues.Then, we down-regulated TMEM16A expression in AGS cells transfected with TMEM16A shRNA for48hours, protein levels of E-Cadherin was significantly increased compare with the control group by Western blot analysis.更多还原