基于GAL-3研究巨噬细胞在肝癌耐药中的作用及调控机制

In tumor microenvironment，Exosomes play a leading role in the communication between Hepatoma cells and macrophages. Data from our previous studies showed that sorafenib resistant hepatoma cells-derived exosomes could promote Galectin-3 (GAL-3) secretion by macrophages, and in turn GAL-3 could enhance the ability of sphere formation by hepatoma cell lines in vitro, and reduce the hepatoma cell’s drug sensitivity, which suggests that GAL-3 secreted by macrophages plays an important role in drug resistance of liver cancer in tumor microenvironment, but the up-regulating mechanism of GAL-3 in macrophages stimulated by exosomes has not been fully elucidated. The purpose of current project is to screen the key component in exosomes which up-regulates macrophages GAL-3 secretion by RNA-Seq, RIP, Co-IP, protein mass spectrometry, and lentivirus packaging and infection system, especially to explore its regulatory mechanism. Meanwhile, research the influence of GAL-3 on stem cell characteristics in hepatoma cells and then enhance the effect of drug resistance of liver cancer. Furthermore, nude mice model and clinical patient serum samples would be involved to check the sensitivity of liver cancer to sorafenib upon treatment with different functional status macrophages. This project could help us understanding the relationship between macrophages and tumor drug resistance in microenvironment, and benefit us to find new clue and strategy in reversing drug resistance of liver cancer.

肿瘤微环境中外泌体是介导肝癌细胞与巨噬细胞间信息传递的重要媒介。申请人前期研究发现，耐药肝癌细胞来源的外泌体可致巨噬细胞半乳糖凝集素-3（GAL-3）分泌增加，GAL-3又致耐药肝癌细胞体外成球能力增强，并使其对药物索拉非尼的敏感性降低。提示，肿瘤微环境中外泌体致巨噬细胞GAL-3的高表达在肝癌耐药中发挥重要作用，但外泌体促进巨噬细胞GAL-3表达上调的机制目前尚未完全阐明。本项目拟通过RNA-Seq、RIP、Co-IP、蛋白质谱、慢病毒包装及感染等手段筛选外泌体中致巨噬细胞GAL-3分泌的关键组分，并研究其调控GAL-3表达的机制，深入探讨GAL-3通过促进肝癌细胞干性的形成进而增强其耐药的作用；进一步利用裸鼠皮下移植瘤模型、临床患者血清样本等手段探讨不同功能状态巨噬细胞对肝癌索拉非尼耐药的影响。本研究可加深理解肿瘤微环境中巨噬细胞与肿瘤耐药的相互关系，为逆转肝癌耐药提供新的策略和启示。

外泌体是肿瘤微环境中介导肿瘤细胞与基质细胞信息交流的重要媒介。本课题主要研究耐药肝癌细胞来源的外泌体通过促进巨噬细胞半乳糖凝集素-3（Galectin-3，GAL-3）的分泌进而增强肝癌耐药的分子机制。申请人通过收集耐药肝癌细胞（HepG2-SR、Huh7-SR）及非耐药肝细胞（HepG2、Huh7）来源的外泌体作用巨噬细胞（MΦ-THP-1），ELISA检测巨噬细胞GAL-3的分泌情况，进一步利用外源表达的GAL-3蛋白作用肝癌细胞，寻找与GAL-3蛋白相互作用的膜分子，探讨GAL-3蛋白对肝癌细胞侵袭转移及干性表型的影响，并阐述其机制。结果证实，耐药肝癌细胞来源的外泌体可明显促进巨噬细胞GAL-3的分泌，GAL-3蛋白通过与CD147和integrinβ相互作用，可活化肝癌细胞MAPK、PI3K/AKT、NF-κB信号通路，致肝癌细胞N Cadherin、MMP9的表达增强，干性表型CD13表达升高。提示耐药肝癌细胞来源的外泌体可使巨噬细胞GAL-3分泌增加，同时GAL-3蛋白又可致肝癌细胞侵袭转移能力增强，干性表型增强。本研究的预期结果可阐明巨噬细胞在肝癌耐药中的作用及机制，为逆转肝癌耐药提供重要启示。