EWS-Fli1融合蛋白对CYR61基因表达的调控及其在尤文肉瘤免疫逃逸中的作用和机制研究

Ewing sarcoma (ES) is one of most frequent primary malignant bone tumors with high incidence secondary to osteosarcoma in childhood and adolescence. Although EWS-Fli1 fusion gene is considered as the crucial oncogene in tumorigenesis of ES, it can't be recognized and killed by immune cells as a kind of tumor antigen. Therefore, it is very important for the application of novel immunotherapeutic strategy in the future to discuss its role in the mechanism of immune escape. To date, some studies have demonstrated that the protective effects of inflammation are a series of local response focused on the vascular alteration. CYR61 is a cysteine-rich secreted protein which can regulate various processes including angiogenesis and inflammation. Our preliminary results showed that Cyr61 was rapidly upregulated and the sequential release of pro-inflammatory productions after knockdown of EWS-Fli1 fusion protein. The purpose of this study is to investigate the mechanism involving CYR61 compromised by EWS-Fli1 using Luciferase and ChIP assay and CYR61-mediated immune responses during inflammation by means of siRNA technology, FACS and NK cells cytotoxity assay in vitro and in vivo, finally elucidating its possible underlying molecular mechanism. The present study might provide new insight into the pathogenesis of ES and development of novel immunotherapeutic medicines against it.

尤文肉瘤是常见的恶性骨肿瘤，发病率在青少年仅次于骨肉瘤。虽然EWS-Fli1融合基因是尤文肉瘤形成的主要致癌基因，但是它作为肿瘤抗原并未受到机体免疫系统的攻击和清除, 因此深入探讨其免疫逃逸机制，对于指导临床建立免疫治疗新策略意义重大。目前研究表明炎症反应的防御作用是以血管系统改变为中心的 一系列局部反应。CYR61是一种富含半胱氨酸的分泌性蛋白，能诱导血管发生，参与炎症反应等过程。我们的前期研究发现EWS-Fli1的表达被下调后早期诱导了CYR61的上调及由此介导的促炎症因子的产生。本次立项研究拟采用荧光素酶报告基因技术，ChIP分析法明确EWS-Fli1对CYR61的调控机制；利用siRNA技术，流式细胞仪和自然杀伤细胞毒性分析通过体外及体内实验探讨CYR61在炎症反应过程中可能产生的免疫激活效应，并明确其作用的分子机制。该研究有望在尤文肉瘤病因学研究及免疫治疗药物开发方面开辟新的视野

EWS-Fli1融合基因作为尤文肉瘤的主要致癌基因,其在机体中的免疫逃逸机制至今尚不清楚。本研究从炎症与免疫之间的相关性入手，在病人标本和细胞株中发现一种能诱导血管发生，参与炎症反应的分泌性蛋白CYR61呈现低表达；并且荧光素酶报告基因技术和ChIP分析法显示EWS-Fli1通过与CYR61启动子ETS位点直接结合从而抑制其转录和表达。另一方面，应用慢病毒感染我们成功筛选了EWS-Fli1去表达和CYR61过表达两种稳定克隆。实时定量PCR，流式细胞仪和自然杀伤细胞毒性分析显示CYR61的上调介导了促炎症因子(TNF-α和IL-1β) 和抗原递呈促进因子 (IL-2, IL-4, IL-6, IL-8, IL-11, IL-17和IFN-γ)的产生；其不但改变了细胞形态和游走能力，而更为重要的是它增加了NK细胞表面上多组织相容性复合物 (MHC)-Ⅰ型链相关蛋白 (MICA/B) 和其受体NKG2D的表达和结合，从而增强了NK细胞对尤文肉瘤细胞的免疫杀伤作用。本研究阐述并验证了EWS-Fli1融合基因通过抑制CYR61介导的炎症-固有免疫连锁反应作用，逃避机体免疫系统监控的假说，并为尤文肉瘤病因学研究及免疫治疗药物开发方面开辟新的思路。