平分型N-糖链在卵巢癌干细胞中的作用和机制研究

Increasing research indicated that epithelial ovarian cancer (EOC) recurrence and poor clinical outcome of cancer patients may be due to the fact that a small subset of stem-like cells, called cancer stem cells (CSCs).Therefore, in order to improve prognosis of ovarian cancer,cancer stem cells represent an attractive therapeutic target for ovarian cancer. Altered glycosylation contribute to cancer development and progress and glycans has been reported as key factors for cancer stem cell relagulation. However, glycosylation structure and function of ovarian cancer stem cell has been rarely reported due to difficulty in obtaining enough ammout of cancer stem cells. Recently, through micro-glycosylation analysis methods developed in our group, we found that bisecting N-glycans was obviously decreased in ovarian cancer stem cell comparing with non cancer stem cell. The ability of cell self-renewal was also affected when the expressing of bisecting N-glycans were changed in ovarian cancer stem cell. In addition, the alteration of bisecting N-glycans was found associated with the activity of Notch signaling pathway.Therefore, all these data indicated that bisecting N-glycans play important role in ovarian cancer stem cell. In this project,we aim to study the function and mechanisms of bisecting N-glycans’s in the ovarian cancer stem cell in vivo and in vitro. This project may lead to insights into the causes and treatment of EOC and can provide potential EOC therapeutic implications for us.

越来越多证据显示卵巢癌易耐药、复发及转移的根源在于卵巢癌干细胞，对其靶向治疗，可望改善卵巢癌高致死率等问题。糖链在肿瘤中发挥重要作用，也被认为是肿瘤干细胞获得特性的关键分子，目前却因很难富集到足够量肿瘤干细胞而使其糖链结构和功能研究难以深入。申请人前期发展的微量糖链鉴定方法，可实现微量肿瘤干细胞糖链结构鉴定，基于此发现了卵巢癌干细胞相比非干细胞平分型N-糖链明显降低，同时发现该糖链改变与Notch分子剪切相关。增加其特异性糖基转移酶Mgat3表达抑制卵巢癌干细胞自我更新，提示该糖链在卵巢癌干细胞特性维持中发挥重要作用。本项目拟利用人卵巢癌组织样本中分选卵巢癌干细胞及Mgat3基因敲除小鼠，通过体内外实验研究平分型N-糖链通过Notch等信号通路调控卵巢癌干细胞的机制，并解析调控过程中该糖链变化结构特征，从糖基化异常的角度阐明卵巢癌干细胞特性维持的分子机制，可望为卵巢癌靶向治疗提供新的思路。

通过本项目研究，提示平分型 N-糖链是卵巢癌干细胞的特征糖链，对卵巢癌干细胞生物特性具有重要影响，基于利用凝集素亲和色谱结合质谱法从卵巢癌干细胞里分离鉴定含有平分型 N-糖链的膜蛋白时，鉴定到剪切 Notch 分子的 γ-分泌酶的关键调控亚基 Nicastrin的发现，我们对Nicastrin的糖基化位点进行了鉴定，通过完整糖肽分析方法鉴定到5个糖基化位点，结合Nicastrin蛋白结构特点，我们发现Nicastrin小亚基上有个可以盖住大亚基上的底物结合位点的“盖子”区域上有两个糖基化位点，Asn55和Asn435，并发现其主要为高甘露糖糖型，提示前期发现的平分型糖型降低可能是因为卵巢癌干细胞中高甘露糖型增多导致；通过对这两个糖基化位点区的单点和双点突变实验，结果发现435位点突变后发现Nicastrin蛋白半衰期缩短，并提示与泛素化作用增强有关，细胞的Notch的剪切体合成减少，下游信号通路的转录因子的表达降低，说明该位点的糖基化修饰影响了Nicastrin蛋白的功能进而影响了卵巢癌细胞的干性。因此，我们的研究提示Nicastrin蛋白435位糖基化修饰在卵巢癌干性维持中具有重要作用，有望为卵巢癌靶向治疗提供潜在的新靶点。