N-乙酰氨基半乳糖转移酶4调控细胞膜表面CD44的糖基化修饰参与肝癌发生发展的分子机制和临床意义研究

Hepatocellular carcinoma (HCC) is the third most common malignancy and the second leading cause of cancer-related death in China. It is urgent to elucidate the exact molecular mechanism of pathogenesis and recurrence of HCC. Since glycosylation plays a pivotal role during tumorigenesis and progression of HCC, establishment of the working model of glycosylation pattern including functional phenotypes and regulatory mechanisms could break a new field for early diagnosis, molecular prognosis and molecular targeted therapy of HCC. N-acetylgalactosaminyltransferase 4 (GALNT4) plays an irreplaceable role in tumorigenesis and tumor progression due to its unique substrates and glycosylation sites. Our preliminary studies showed that the expression level of GALNT4 in tumor tissues was significantly decreased than peri-tumor tissues surgically resected from patients with HCC, which associated with patient survival. Furthermore, ectopic expression of GALNT4 could inhibit migration, invasion and spheroids formation of hepatoma cells. Based on the preliminary data, the current project proposal is designed to illuminate the molecular mechanisms, functional significance and interventive strategies of aberrant glycosylation including the activation of miR-9/GALNT4/CD44 signaling during hepatocarcinogenesis. Human hepatoma cells and tumor specimens surgically resected from patients with HCC will be used in our attempts to establish the model of molecular regulation, preoperative diagnosis, postoperative survival assessment and interventive therapy based on the glycosylation pattern in HCC, which could help us lay the theoretical foundation for the development of diagnostic reagents and targeted therapeutic drugs.

近年来，我国肝细胞癌发生率与死亡率始终居高不下，针对其发病、转移、复发的相关研究亟待加深与突破。蛋白质糖基化修饰在肝癌发生发展过程中扮演重要角色，N-乙酰氨基半乳糖转移酶4（GALNT4）因其独特的糖基化修饰靶点而在肿瘤发生发展过程中发挥关键作用。在前期研究中，我们发现肝癌患者癌组织中的GALNT4表达水平显著低于癌旁组织，并与患者生存显著相关。本申请项目拟在前期研究基础上，以人肝癌组织及肝癌细胞系为研究对象，围绕蛋白质糖基化修饰调控肝癌发生发展的分子机制及其干预策略这一关键科学问题，深入分析肝癌发生发展过程中miR-9/GALNT4/CD44信号活化的调控机制及功能意义，初步建立蛋白质糖基化修饰调控肝癌发生发展的分子机制、术前诊断、术后生存评估和干预治疗模型，为开发基于蛋白质糖基化的诊断试剂和靶向治疗药物奠定理论基础。

N-乙酰半乳糖氨基转移酶家族(GALNTs)参与Mucin样O-糖基化的起始步骤，其表达失调可能产生异常的、截短的O-糖基，从而在恶性转化过程中发挥关键作用。GALNT4是少数几种倾向于催化部分GalNAc-糖基化底物并修饰其他已知GALNTs未利用的位点的亚型之一。本研究旨在评估GALNT4表达对肝细胞癌(HCC)恶性转化的影响。进行免疫组织化学和原位杂交分析以分别评估临床标本中GALNT4和miR-9水平。GALNT4的表达在原发性HCC组织中被显著抑制，而GALNT4的表达降低与HCC患者的不良生存密切相关。功能研究表明，抑制GALNT4可促进肝癌细胞的迁移、侵袭、耐药、干细胞特性以及体内肿瘤生长。野生型GALNT4可以修饰表皮因子生长受体(Epidermal Growth Factor Receptor, EGFR)上的O-连接糖基化，从而调节EGFR的活性。荧光素酶活性分析进一步确定microRNA-9 (miR-9)在HCC细胞GALNT4的表达中发挥了关键作用。此外，恢复GALNT4表达会减弱miR-9介导的致癌功能。Kaplan-Meier生存分析表明，miR-9/GALNT4表达特征对改善HCC患者的风险分层具有重要的预后意义。总之，本研究建立了miR-9/GALNT4轴作为HCC患者潜在的不良预后因素和治疗靶点，同时也为开发基于蛋白质糖基化的诊断试剂和靶向治疗药物奠定理论基础。