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。

N-乙酰氨基葡萄糖转移酶Ⅴ（GnT-Ⅴ）是糖蛋白上 N-糖链分支结构形成的关键酶，其表达与肿瘤的发生、发展密切相关。然而，GnT-Ⅴ与膀胱癌化疗药物敏感性的关系尚未阐明。本研究发现，与正常膀胱癌旁组织比较，GnT-Ⅴ在膀胱癌组织和细胞中表达异常增高，且与膀胱癌的病理分期与淋巴组织浸润呈正相关。体外研究发现，下调GnT-Ⅴ能够引起膀胱癌细胞周期阻滞，促进其凋亡，且能明显提高膀胱癌细胞对抗癌药物吉西他滨的敏感性。荷瘤小鼠体内研究，我们发现与吉西他滨单独用药比较，GnT-Ⅴ沉默联合吉西他滨治疗能够明显抑制肿瘤细胞的增殖、肿瘤血管生成并延长荷瘤小鼠的生存期；进一步研究发现， GnT-Ⅴ沉默引起化疗药物吉西他滨转运载体hENT1（人平衡型核苷转运蛋白1）上β1,6 GlcNAc糖链分支减少，从而导致转运进入膀胱癌细胞内的吉西他滨浓度升高。提示：下调GnT-Ⅴ可能是通过引起hENT1的N糖基化改变，进而改变其转运活性，从而提高膀胱癌细胞对化疗药物吉西他滨的敏感性。本研究将为膀胱癌患者术后系统化疗方案的建立提供实验和理论依据，同时对指导个体化临床化疗用药具有重要意义。