膜联蛋白A2亚硝基化诱导肝细胞癌上皮-间质转化的机制

Annexin A2 (ANXA2) plays an important role in the Epithelial-mesenchymal transition (EMT) of hepatocellular carcinoma (HCC), but its mechanism is still obscure up to now. On the basis of our previous finding that ANXA2 promotes HCC metastasis, in HCC cells, we intend to explore if ANXA2 S-nitrosylation is iNOS-dependent, illuminate the mechanism that ANXA2 S-nitrosylation promotes itself cytomembrane transfer and heterotetramer formation, subsequently induces Rho-dependent EMT via twist/miR-10b/Snail1 pathway mediated by the c-Src S-nitrosylation, analyze the effect of ANXA2 S-nitrosylation on invasion and migration potential of HCC cells. HEK293 cells were transfected with track-cmv-RFP-ANXA2 wild type plasmids or mutant plasmids and incubated with GSNO, the differences of ANXA2 S-nitrosylation levels between groups were compared so as to identify the key S-nitrosylation site of ANXA2. After building the metastatic model of human liver cancer in nude mice, we plan to analyse the expression differences of EMT- associated molecules between ANXA2 S-nitrosylation or not, compare the degree differences of HCC metastasis by small animals living imaging system. This study aims to illuminate the novel mechanism of ANXA2 S-nitrosylation inducing EMT of HCC cells and provide new biological therapeutic target for HCC metastasis.

膜联蛋白A2（Annexin A2，ANXA2）在肝细胞癌的上皮间质转化（EMT）过程中发挥关键作用，但机制尚不清楚。在前期发现ANXA2促进肝细胞癌转移的基础上，本项目拟在肝细胞癌细胞上，探讨ANXA2亚硝基化对iNOS依赖性；阐明ANXA2亚硝基化促使其膜定位和异四聚体形成，进而通过c-Src亚硝基化介导的twist/miR-10b/Snail1通路实现Rho依赖型EMT的新机制；分析ANXA2亚硝基化对肝癌细胞侵袭迁移潜能的影响。在HEK293细胞上分别转染ANXA2野生型和突变体质粒，GSNO孵育，比较ANXA2亚硝基化水平差异，以鉴定ANXA2亚硝基化位点。构建人肝癌裸鼠转移模型，比较瘤体中EMT相关分子在ANXA2亚硝基化前后的表达差异，用小动物活体成像系统比较肝癌转移程度差异。本研究旨在阐明ANXA2亚硝基化诱导肝细胞癌发生EMT的新机制，为肝细胞癌转移的生物治疗提供新靶点。

肝癌转移是其死亡率居高不下的关键原因，包括氧化还原反应在内的肿瘤微环境在肝癌转移中的作用机制尚不明确。本研究阐明了肝癌细胞上亚硝基化的膜联蛋白A2(ANXA2-SNO)促进Snail1核定位及其乙酰化，招募肿瘤相关巨噬细胞并促进巨噬细胞中iNOS的活化、释放NO，从而进一步促进肝癌细胞ANXA2-SNO水平。与此同时，ANXA2-SNO促进了肝细胞癌细胞的EMT，进而促进了肝癌细胞的侵袭和迁移。肝癌组织中ANXA2-SNO水平与肿瘤浸润巨噬细胞（tumor-associated macrophage, TAM）呈现正相关性，根据多因素分析，ANXA2-SNO是一个独立预后因子，ANXA2-SNO高水平意味着患者更早的死亡和复发。进一步分析发现，ANXA2-SNO有助于TNM 3期4期患者总生存期和无复发生存期的分层，与TNM协同作用，有助于提高患者的准确性和灵敏度。在病理分级方面也有类似发现。诺曼图模型显示，包括ANXA2-SNO水平、TAM浸润程度的诺曼图模型对于OS、DFS的预测准确性都明显高于TNM模型。总之，本研究阐明了ANXA2-SNO通过招募肿瘤相关巨噬细胞促进肝癌细胞EMT的机制，以及ANXA2-SNO的临床价值，从而为肝细胞癌转移的生物治疗提供新靶点和新的治疗思路，为肝癌手术治疗、术后辅助化疗患者以后提供指导方案。