GnT-Ⅴ的表达及转运蛋白N-糖基化对膀胱癌吉西他滨化疗敏感性的影响

hENT1 and P-gP, two anti-tumor drug transporters, are N-glycosylated glycoproteins. N-acetylglucosaminyltransferase Ⅴ (GnT-Ⅴ) is a key enzyme which catalyzes the formation of branching structures of N-glycans on the glycoproteins. We noticed that GnT-Ⅴexpression increased significantly in human bladder cancer tissues and cells compared with the normal tissues; and downregulation of GnT-Ⅴ significantly improved the sensitivity to anti-tumor drug gemcitabine in bladder cancer cells. Therefore, we presume that chemosensitivity may be closely associated with the N-glycosylation of drug transportor in bladder cancer cells, but the molecular mechanism is still unknown. In this study, we’ll analyze associations of GnT-Ⅴ expression with clinical-pathological factors and chemosensitivity to gemcitabine by the detection of GnT-Ⅴ expression in clinical pathological tissue specimens combine with the retrospective study on corresponding clinical cases. Moreover, we will investigate the effect of GnT-Ⅴon chemosensitivity to gemcitabine, the expression and N-glycan branches of anti-tumor drug transports and the transport activity both in vitro and in vivo via gene silencing and rescue combine with animal tumor xenografts. In addition, we’ll reveal the effect of N-glycosylation on the transport activity of hENT1 and P-gP through N-glycosylation site mutant study. This study was designed to propose and reveal the mechanism of the effect of N-glycosylation on the chemosensitivity in bladder cancer and to provide a new idea of individualized chemotherapy for patients with cancer.

药物转运蛋白hENT1和P-gP是N-糖基化的糖蛋白。N-乙酰氨基葡萄糖转移酶Ⅴ（GnT-Ⅴ）是催化糖蛋白上N-糖链分支形成的关键酶。申请者观察到，与正常相比，人膀胱癌组织和细胞中GnT-Ⅴ表达显著升高；下调其表达明显提高膀胱癌细胞对化疗药物吉西他滨的敏感性，因此推测：膀胱癌化疗药物敏感性可能与药物转运蛋白的N-糖基化密切相关，但分子机制不明。本课题1）通过临床病理组织和病例资料的回顾性分析，研究膀胱癌中GnT-Ⅴ表达的临床病理意义及与吉西他滨敏感性的关系；2）通过基因沉默及表达重获、体内动物荷瘤模型构建，在体内外观察GnT-Ⅴ表达对膀胱癌细胞吉西他滨敏感性、对药物转运蛋白的表达、N-糖链分支结构及转运活性的影响；3）通过hENT1和P-gP的N-糖基化位点突变，观察N-糖基化对其转运活性的影响。本研究旨在提出并阐释N-糖基化影响膀胱癌化疗药物敏感性的机制，为肿瘤的个体化治疗提供新思路。