NOTCH1(C1133Y)突变通过调控FBXW7在口腔癌发生、发展中的作用

Our previous studies showed that the mutation rate of NOTCH1 gene in oral squamous cell carcinoma (OSCC) was as high as 40%, but the function of these mutations was not clear. One of the hot spot mutations in C1133Y has attracted our attention because of the destructive nature of bioinformatics. It was found that the mutation significantly promoted the proliferation and invasion of oral cancer cells. Further studies showed that mutant NOTCH1 protein existed in the form of full length in the endoplasmic reticulum, and combined with the the ubiquitin ligase FBXW7 in endoplasmic reticulum leading to the degradation of FBXW7, thereby increasing the level of FBXW7 substrate like c-Myc. Based on the previous research, we will perform the construction of mutant and wild-type NOTCH1 expression vector, the transfection of oral cancer cell lines, and will use CRISPR/cas9、laser confocal microscope and Co-IP methods to study deeply the effects of mutations on the proliferation and invasion of oral squamous cell carcinoma both in vitro and in vivo; On mechanism, FBXW7 degradation after combination with NOTCH1 (C1133Y) may play an important role in the occurrence and progression of oral cancer. The results of this study will elucidate the effects of NOTCH1 mutation on NOTCH pathway and oral cancer from a new perspective.

我们前期研究显示NOTCH1基因在口腔鳞状细胞癌（口腔鳞癌）中的突变率高达40%，但对于这些突变的功能尚不清楚。其中一个热点突变C1133Y因生物信息学提示极具破坏性引起我们注意。预实验发现该突变显著促进口腔癌细胞的增殖与侵袭。进一步研究发现突变的NOTCH1蛋白以全长形式存在于内质网中，且与内质网中的泛素连接酶FBXW7结合并导致其降解，由此增加了FBXW7的底物c-Myc等的水平。本项目拟在前期研究结果的基础上构建突变与野生型NOTCH1表达载体，转染口腔癌细胞系，使用CRISPR/cas9、激光共聚焦、Co-IP等方法在体外和动物实验两个层面深入研究该突变对口腔鳞癌增殖及侵袭的影响；并以FBXW7为机制上的突破点，研究NOTCH1(C1133Y)对于口腔癌发生、发展的作用。研究结果将从全新的角度阐明NOTCH1突变对NOTCH通路及口腔癌的影响。

本课题组先前发现在中国口腔鳞癌患者中NOTCH1Abruptex结构域突变最多，其中包括一个热点突变(C1133Y)。FBXW7β是一种E3泛素连接酶，通过降解调节包括NOTCH1在内的蛋白质网络。为进一步揭示NOTCH1基因与FBXW7β的关系，我们设计并开展了此项研究。.实验结果显示：1.通过定点诱变，发现NOTCH1(C1133Y)突变型能促进增殖、迁移与侵袭。激光共聚焦显示NOTCH1(C1133Y)突变后与内质网特异性标志物calnexin共定位。2.利用RT-PCR和蛋白印迹分析口腔鳞癌与癌旁组织中FBXW7β的表达。体外实验发现，过表达FBXW7β显著抑制了细胞的生长与侵袭。沉默FBXW7β后，极大地促进了细胞生长。.获得表型效应后，我们进行了机制探索1.通过过表达FBXW7β与NOTCH1(C1133Y)，发现FBXW7β调控NOTCH1(C1133Y)诱导的致癌表型改变。FBXW7β在NOTCH1(C1133Y)突变激活的Akt/ERK/NFκB的通路中发挥重要作用。2.通过免疫共沉淀发现，FBXW7β通过泛素化介导NOTCH1(C1133Y)的降解。.由于在中国人中已检测到NOTCH1基因的全面突变，新发现的FBXW7β与NOTCH1(C1133Y)之间的相互作用可能为研究NOTCH1在口腔鳞癌细胞中的降解，特别是具有Abruptex结构域突变的口腔鳞癌细胞提供了一个新的视角，并为口腔鳞癌的治疗提供了一个潜在的靶点。