潜在癌基因14-3-3β对胶质瘤生物学特性的影响及其配体分子的鉴定

The prognosis of glioma is dismal, despite of a variety of conventional therapies including radiotherapy, chemotherapy and surgery. So it is important to find a new treatment for gliomas. Our previous study has shown that one subtype of 14-3-3, 14-3-3β is overexpressed in gliomas and its expression is increased markedly with an increase in the pathologic grade of human gliomas; whereas 14-3-3β immunoreactivity was not seen in glial cells from normal brain tissue. Morever, the patients with higher expression level of 14-3-3β had significant short survival time compared to those with lower expression of 14-3-3β. 14-3-3 proteins, potentially acting both as oncogenes and as tumor suppressors, can bind to a wide variety of proteins involved in signal transduction, cell cycle control, vesicular transport, DNA replication and apoptosis. Based on the previous results we speculate that 14-3-3β may function as a potential oncogene for the tumorigenesis of glioma and plays a key role in the malignant transformation of glioma. Therefore, the current study will use the yeast two-hybrid and co-immunoprecipitation techniques to obtain the functional proteins that interact with 14-3-3β in order to further understand the mechanism underlying the tumorigenesis role of 14-3-3β, and the techniques of gene silencing and Western blotting will be applied to explore the relationship of 14-3-3β and glioma on both glioma cell lines and tumor tissue samples. Furthermore, through methods such as immunohistochemistry combined with follow-up data from patients, to further explore the 14-3-3β and its ligand expression level with pathological grade and prognosis, and provide potential target for the molecular therapy of this devastating disease.

胶质瘤患者即使术后接受放、化疗等综合治疗，疗效依然欠佳。曹卫东发现，14-3-3蛋白在胶质瘤中的表达强度和范围有随着肿瘤恶性程度的增高而升高。本课题组前期研究发现14-3-3β在正常脑组织中的胶质细胞中未见表达，但是随着肿瘤级别的增高，表达更为明显，且β亚型高表达的胶质瘤患者生存期明显低于低表达患者。由此我们推测14-3-3β可能为胶质瘤发生的潜在癌基因，有可能成为胶质瘤治疗的新靶点。本课题拟以肿瘤组织和胶质瘤细胞系为研究对象，通过酵母双杂交等方法等筛选胶质瘤主要信号通路中可能与14-3-3β相互作用的配体分子，初步探讨14-3-3β促进胶质瘤发生发展过程中涉及的信号通路；通过RNAi等方法探讨14-3-3β及其配体分子对胶质瘤生物学特性的影响；通过免疫组化等方法并结合患者的随访资料，进一步探讨14-3-3β及配体分子表达水平与病理分级和预后的关系，为胶质瘤的防治及靶向治疗提供科学依据。

胶质瘤患者即使术后接受放、化疗等综合治疗，疗效依然欠佳。本课题在前期研究的基础上，推测14-3-3β可能是胶质瘤发生的潜在癌基因，有可能成为胶质瘤治疗的新靶点。本课题以肿瘤组织和胶质瘤细胞系为研究对象：（1）通过酵母双杂交及免疫共沉淀从正反两方向都证实了14-3-3β与FBI1的相互作用。（2）采用免疫组化ABC法检测发现在正常脑组织中，FBI1主要表达于神经元，但在人脑胶质瘤组织中，FBI1的免疫反应评分随着肿瘤的级别增高而增高。（3）WB检测提示在U87细胞系中14-3-3β高表达，而在正常胶质细胞系SVGp12中未见表达。为了明确14-3-3β在胶质瘤中的作用，我们通过向U87细胞系转染Si-14-3-3β，与对照组相比Si-14-3-3β组中14-3-3β的表达在转染后显著降低，且细胞活性降低。（4）为了证实14-3-3β敲除后的抑癌效应是否与凋亡有关，U87转染Si-14-3-3β后，通过流式细胞术检测提示14-3-3β敲除后显著提高U87细胞的凋亡率，并且显著抑制细胞色素C的释放。（5）敲除14-3-3β后，通过ER追踪染色观察， ER的荧光强度增加，且Ca2+从ER释放，Si-14-3-3β转染显著抑制XBP1S mRNA的表达。为了进一步明确ERS，我们通过WB检测转染Si-14-3-3β后CHOP和Caspase-4的表达情况。结果提示：与对照组相比，CHOP和Caspase-4表达增加。（6）为了探索潜在的分子机制，我们检测转染后U87细胞系中β-catenin的表达，下调14-3-3β显著降低β-catenin的核定位和降低Topflash的活性，其活性与CHOP呈负相关。Si-CHOP转染可以阻止Si-14-3-3β诱导的Topflash活性降低和细胞活性的下降，结果提示Wnt有可能是CHOP的靶点。此外，为了探索Wnt在Si-14-3-3β诱导的抑癌效应中的作用，sFRP通常用来抑制Wnt，结果提示，sFRP和Si-14-3-3β在Wnt激活和细胞存活率方面没有额外的影响。Si-CHOP和sFRP分别用来抑制CHOP和Wnt，在Si-CHOP转染的U87中，sFRP和Si-14-3-3β在Wnt激活和细胞存活率方面没有额外的影响。（7）在体内实验中，我们通过下调14-3-3β在胶质瘤细胞系U87中的表达，建立人脑胶质瘤裸鼠皮下移植瘤模型。定