4.1G对恶性外周神经鞘瘤的抑制作用及机制研究

Malignant peripheral nerve sheath tumors(MPNSTs) are mostly high-grade sarcomas which derive from Schwann cell. Our pilot experiment demonstrated that, compared with the strong expression of 4.1G on normal Schwann cells, its expression decreased in neurofibroma,and even none expression could be detected in some MPNSTs. This kind of expression pattern suggests that 4.1G might function as a suppressor during MPNST tumorigenesis. The 4.1 family proteins are adaptors linking transmembrane proteins to cortical actin cytoskeleton, more and more evidence have demonstrated that they could inhibit the tumor cell proliferation in recent years. Considering the activation of transmembrane tyrosine receptor EGFR signalling pathway in MPNST, we hypothesize that,as a linker between membrane proteins and cytoskeleton, 4.1G might inhibit the proliferation of MPNST through regulating the activation of membrane receptor EGFR pathway. Therefore, we design the following project to verify our hypothesis. Firstly, we will set up the 4.1G exogeneous overexpression and 4.1G knockdown cell models using human MPNST cell lines. The effect of 4.1G on cell proliferation will be tested by cell counting method. And the regulating mechanism to cell proliferation will be confirmed by flowcytometry cell cycle analysis and Annexin V apoptosis analysis. Secondly, we will check the possibility that the proliferation regulating function of 4.1G is mediated by EGFR signaling pathway. Expression of EGFR/pEGFR and its downstream MAPK/ERK and PI3K/Akt and their phosphated forms will be measured on the 4.1G overexpression/knockdown cells. In addition, EGF or EGF with EGF inhibitor treatment to cells will be performed to confirm the EGFR pathway involvement. Thirdly, Texas red-EGF fluorescence tracing and biotin labelling of surface proteins are planed to explore the possibile changes of the receptor endocytic trafficking process caused by 4.1G overexpression/knockdown. Lastly, 4.1G protein expression profile on human MPNST and neurofibroma tumor samples will be checked, and the relationship between 4.1G expression and prognosis of these cases will be analysed to confirm the tumor suppressor function of 4.1G to MPNST. Through this project, we hope to clarify the clinical significance of 4.1G to MPNST tumorigenesis and prognosis, and know more about the regulation mechanism of EGFR signaling to direct the use of EGFR target therapy in future.

恶性外周神经鞘瘤（MPNST）多是高度恶性肉瘤。我们前期发现，4.1蛋白中的4.1G在正常施万细胞中高表达，但在MPNST中表达下降甚至缺失。由于4.1是膜蛋白和皮层细胞骨架间的桥梁，而MPNST中膜受体EGFR通路异常活化，并且其它4.1蛋白能抑制细胞增殖，因此推测4.1G通过调节EGFR抑制MPNST的增殖，发挥抑癌基因作用。本课题拟首先利用人MPNST细胞系，以4.1G过表达、敲减/逆转及裸鼠坐骨神经原位种植成瘤试验，验证4.1G对MPNST增殖的抑制；然后以EGF/+EGFR抑制剂处理，研究4.1G对EGFR及下游MAPK/ERK和PI3K/Akt通路的调节；以荧光标记EGF追踪和膜蛋白生物素标记法，初步探索4.1G通过受体内吞转运调节EGFR通路的分子机制；以人实体肿瘤标本验证4.1G的抑癌作用。以期阐明4.1G表达对MPNST预后的临床意义，为指导EGFR靶向治疗提供理论基础。

我们前期研究发现，4.1G蛋白在正常外周神经施万细胞中高表达，但在外周神经肿瘤良性神经纤维瘤和恶性外周神经鞘瘤（MPNST）中表达下降，部分MPNST中表达缺失，提示4.1G可能是MPNST的抑癌基因。本课题对于4.1G蛋白在施万细胞肿瘤发生和发展进程中的作用和调控进行了有意义的初步探索。.本课题首先进行细胞培养体外研究。采用ATCC细胞库的细胞系为体外研究对象。包括人MPNST细胞系sNF96.2、sNF02.2和永生化的正常施万细胞RT4，以人正常外周神经组织为对照。免疫印迹和免疫荧光染色法在蛋白水平上证实正常的RT4细胞和恶性MPNST细胞在4.1G表达水平和4.1G蛋白亚细胞定位方面存在差异。并且，同样是MPNST细胞系，局部复发的96.2细胞和发生肺转移的02.2细胞，其生长速度、细胞形态存在显著不同。经过基因克隆我们发现，从正常人外周神经中克隆出的4.1G异构体与从96.2细胞中克隆出的异构体基本相似，但是发生肺转移的02.2细胞中表达较短剪切体。经过构建不同异构体的真核表达载体，证实不同异构体的亚细胞定位显著不同，提示其对于细胞膜蛋白、细胞膜相关调控分子的稳定、细胞分裂、增殖等发面发挥重要作用。本研究在如下方面有重要意义：转移能力不同的MPNST细胞系表达4.1G异构体不同，提示其既有抑癌基因作用又有促癌基因作用。此方面研究尚未见国内和国际报道。进一步研究较短异构体促进细胞侵袭转移的机制将对未来恶性肿瘤的治疗提供重要靶点。研究较长异构体对细胞转移能力的抑制则可能为MPNST转移生物学行为的预测、膜蛋白及受体调控、膜蛋白相关靶向治疗提供重要根据。