【作者】 罗涛；

【导师】 李新钢；

【作者基本信息】 山东大学 ， 外科学， 2021， 硕士

【摘要】 目的人脑胶质瘤(脑胶质细胞瘤)是中枢神经系统肿瘤中最常见的和侵袭性最高的恶性脑肿瘤,它是由于大脑和脊髓的胶质细胞发生癌变后产生的,因为其具有高度的侵袭性和耐药性,预后极差,所以人脑胶质瘤成为致死性最高的恶性肿瘤之一。人脑胶质母细胞瘤(Glioblastomas,GBM)大约占原发性恶性脑肿瘤的75%,总的发病年龄高峰在30-40岁,胶质瘤复发率比较高,5年的生存率较低。现在对于脑胶质瘤的治疗采取标准化治疗,包括手术、放疗和化疗,虽然采取了多模式治疗方案,有的地方甚至增加了磁场治疗,但是恶性胶质瘤患者在确诊后平均生存时间仅为12-15个月。因此,迫切需要新的分子生物标志物进行诊断和靶向治疗。Hippo信号失调已被发现是包括神经胶质瘤在内的许多癌症发展的基础,这表明该通路可能是人类疾病的一个有吸引力的治疗靶点。在我们之前的研究中,我们发现前列腺跨膜蛋白、雄激素诱导1(PMEPA1)、泛素特异性蛋白酶39(USP39)和肌动蛋白样6A(ACTL6A)通过调控Hippo信号在人脑胶质瘤的进展中发挥重要作用。其他研究表明,一些细胞骨架相关蛋白调节Hippo信号,以控制细胞命运决定和调节细胞生长。细胞骨架相关蛋白 4(Cytoskeleton-associated protein 4,CKAP4)是一种Ⅱ型跨膜蛋白,最初在内质网中发现,但它也与微管结合。后来在血管平滑肌细胞、膀胱上皮细胞和Ⅱ型肺细胞的细胞表面膜中发现了它,它作为多种配体的受体,包括表面活性剂蛋白a(SP-A)、组织纤溶酶原激活剂(TPA)、抗增殖因子(APF)和Dickkopf1(DKK1)蛋白。研究表明,CKAP4在肺癌、肝细胞癌、胰腺癌、食管癌和口腔鳞状细胞癌等多种癌症的发生发展中起重要作用。然而,CKAP4在人类胶质瘤发展中的作用仍是未知的。在本研究中,我们尝试探究CKAP4在人脑胶质瘤中表达情况,并作为致癌基因通过抑制Hippo信号通路参与胶质瘤的恶性进展,以此开发新的分子生物标志物进行诊断和靶向治疗。材料和方法首先,使用GEPIA、CCGA等公共数据库来评价CKAP4在不同级别的脑胶质瘤中的表达情况,再用胶质瘤标本做IHC、western、PCR实验,验证数据库中的结果;接着,敲减胶质瘤细胞中CKAP4的表达水平,观察对肿瘤细胞的增殖、侵袭和迁移的影响,然后再过表达胶质瘤细胞CKAP4的水平,验证对肿瘤细胞功能的影响;之后,通过建立肿瘤颅内原位表达模型,观察敲减和过表达CKAP4后,肿瘤在体内的影响;最后,找出CKAP4通过何种通路来影响肿瘤的功能,以此研发出能够治疗脑胶质瘤的特异性药物。结果CKAP4在人脑胶质瘤中的表达水平升高,其表达增加与肿瘤分级增加相关,而且高表达CKAP4的肿瘤与患者生存期差相关。在GBM细胞系LN229和U251中敲除CKAP4抑制了体外和小鼠原位脑瘤模型的增殖。相反,在体外和体内模型中,过表达的CKAP4增强了 U87MG的这些恶性特性。通过YAP/TAZ启动子控制下荧光素酶报告基因构建的功能分析,CKAP4的表达增加导致了 yes相关蛋白(YAP)/PDZ结合基序(TAZ)和TEAD依赖转录复合物(Hippo信号下游靶点)的激活。结论CKAP4可能作为一种致癌基因,通过抑制Hippo信号,促进胶质瘤的发展。更多还原

【Abstract】 ObjectivesGliomas are the most common and aggressive malignant brain tumors and are associated with high mortality and incidence in humans.Glioblastomas(GBM)account for almost 75%of malignant primary brain tumors and are characterized by a low five-year survival rate and high recurrence.Despite rigorous multi-modal therapy,including surgery,chemotherapy and radiotherapy,patients with malignant glioma survive an average of 12-15 months following primary diagnosis.Therefore,new molecular biomarkers are urgently needed for diagnosis and targeted therapy.Dysregulated Hippo signaling has been found to underlie the development of many cancers,including gliomas,indicating that this pathway may be an attractive therapeutic target for the disease in humans.In our previous studies,we found that prostate transmembrane protein,androgen induced 1(PMEPA1),ubiquitin specific protease 39(USP39)and actin-like 6A(ACTL6A)play a vital role in the progression of human gliomas through regulation of Hippo signaling.Other studies have demonstrated that some cytoskeleton-associated proteins modulate Hippo signaling to control cell fate decisions and regulate cell growth.Cytoskeleton-associated protein 4(CKAP4)is a type II transmembrane protein originally observed in the ER,but it also binds to microtubules.It has since been found in the cell surface membrane of vascular smooth muscle cells,bladder epithelial cells,and type Ⅱ pneumocytes,where it functions as a receptor for several ligands,including surfactant protein A(SP-A),tissue plasminogen activator(TPA),anti-proliferating factor(APF),and Dickkopf1(DKK1)protein.Studies have shown that CKAP4 plays an important role in the development of several types of cancer,including lung cancer,hepatocellular carcinoma,pancreatic cancer,esophageal cancer and oral squamous cell carcinoma.However,the role of CKAP4 in the development of human glioma remains unknown.In this study,we demonstrate that CKAP4 is upregulated in human gliomas and contributes to malignant progression of gliomas as an oncogene through suppression of Hippo signaling.Materials and MethodsFirstly,GEPIA,TOGA,CCGA and other public databases were used to evaluate the expression of CKAP4 in gliomas of different grades.Next,IHC,Western and PCR experiments were performed on glioma specimens to verify the results in the database.Furthermore,CKAP4 expression level in glioma cells was knocked down to observe the effect on the proliferation,invasion and migration of tumor cells.Then,CKAP4 expression level in glioma cells was overexpressed to verify the effect on the function of tumor cells.Then,the effect of CKAP4 knockdown and overexpression on tumor in vivo was observed by establishing tumor in situ expression model.Finally,to find out the pathway through which CKAP4 affects tumor function,so as to develop specific drugs for the treatment of glioma.ResultsThe expression level of CKAP4 in human glioma is increased compared with normal brain tissue,which is correlated with the increase of tumor grade.And the tumor with high expression of CKAP4 is associated with poor survival of patients.CKAP4 knockout in GBM cell lines,LN229 and U251,inhibited the proliferation of gliomas in vitro and mouse brain tumor models in situ.In contrast,overexpression of CKAP4 enhanced these malignant properties of U87MG in vitro and in vivo models.By functional analysis of luciferase reporter gene construction under the control of YAP/TaZ promoter,increased CKAP4 expression leads to activation of YAP/PDZ-binding motif(TaZ)and TEAD-dependent transcription complex(downstream target of Hippo signaling).ConclusionCKAP4 may act as an oncogene and promote the development of glioma by inhibiting Hippo signaling.更多还原