携miRNA-210外泌体介导的缺氧-常氧肝癌细胞间信息传递调控细胞自噬并诱导索拉非尼耐药的机制研究

Sorafenib is fairly effective in the treatment of advanced hepatocellular carcinoma (HCC) while its effectiveness is limited by the hypoxia-induced drug-resistance, and the mechanism of how normoxia tumor cells which are not directly affected by oxygen-deficient environment get drug-resistance is still unclear. Latest research reveals that exosome-induced information transfer between the hypoxic-normoxic tumor cells plays a key role in tumor progress under hypoxic conditions. Our preliminary experiment showed that after co-culture of the exosomes secreted by hypoxic HCC cells with normoxic HCC cells, the sorafenib resistance of the latter went up. Further study showed the hypoxic HCC cells-secreted exosomes secreted were abundant in microRNA-210 which was closely related with autophagy regulation. Thus we hypothesize that hypoxia-stimulated hypoxic hepatocarcinoma cells secrete exosomes around them, then these exosomes carrying miR-210 signaling are absorbed by surrounding normoxic tumor cells and lead to autophagy and then the following sorafenib resistance. In this study, we will establish a co-culture system of hypoxic HCC cells-secreted exosomes and normoxic HCC cells and the nude mice tumor transplant model. With the help of gene interference, autophagy regulation and animal experiments, this study is expected to elucidate the mechanism of exosome inducing information transfer between hypoxic-normoxic HCCs and leading to the progress of the sorafenib resistance, which will provide an experimental basis for finding a new target to prevent and treat sorafenib resistance.

索拉非尼治疗晚期肝细胞性肝癌效果显著，但因缺氧引起的肿瘤细胞广泛耐药限制了疗效的发挥，未受缺氧环境直接影响的正常氧肿瘤细胞如何产生耐药，其机制并不清楚。新近研究提示外泌体介导的缺氧-常氧细胞间信号传递是缺氧环境下肿瘤进展的关键机制，预实验证明将缺氧肝癌细胞分泌的外泌体与常氧肝癌细胞共培养可以提高后者索拉非尼耐药性，进一步研究发现外泌体高表达microRNA-210且此miRNA与自噬调控相关。故提出假设：缺氧刺激后缺氧肝癌细胞向其周围分泌外泌体，这些携带miR-210信号的外泌体被周围常氧肿瘤细胞接收并调控其细胞自噬进而引起索拉非尼耐药。本课题拟建立缺氧肝癌细胞源外泌体与常氧肝癌细胞共培养体系以及裸鼠成瘤用药模型，采用基因干扰、自噬调控及动物实验等方法，阐明外泌体介导缺氧-常氧肝癌细胞间信息传递并影响索拉非尼耐药进程的分子机制，为寻找防治索拉非尼肝癌耐药新靶点提供实验依据。

索拉非尼治疗晚期肝细胞性肝癌效果显著，但因缺氧引起的肿瘤细胞广泛耐药限制了疗效的发挥，未受缺氧环境直接影响的正常氧肿瘤细胞如何产生耐药，其机制并不清楚。我们认为缺氧刺激后缺氧肝癌细胞向其周围分泌外泌体，这些携带miRNA信号的外泌体被周围常氧肿瘤细胞接收，促进肿瘤恶性能力增加并最终引起了索拉非尼耐药。本研究通过建立缺氧肝癌细胞源外泌体与常氧肝癌细胞共培养体系，采用基因干扰、自噬调控等方法，探讨外泌体介导缺氧-常氧肝癌细胞间信息传递并影响肿瘤进展及索拉非尼耐药进程的分子机制。主要有以下重要发现：.1） 肝癌细胞源缺氧外泌体能够增强常氧培养条件下肝癌细胞的增殖、侵袭、迁移、EMT以及索拉非尼耐药等恶性能力。.2）外泌体中的miR1273f，而非课题预设的靶点miR-210，是导致肝癌缺氧外泌体诱导肿瘤增殖、侵袭迁移以及EMT的最关键分子。.3） LHX6/Wnt/β-catenin信号通路可能是miR1273f介导肝癌缺氧外泌体促肿瘤恶性进展的具体分子机制。.4）细胞自噬作为细胞缺氧的下游机制，介导了肝癌细胞源缺氧外泌体促索拉非尼敏感性下降的作用。.5）外泌体中异常表达的miRNA信号以及常氧靶细胞中异常表达的通路信号可能是诱导细胞自噬、介导肝癌细胞源缺氧外泌体促索拉非尼敏感性下降的关键机制。.综上，本项目通过一系列实验证实缺氧肝癌细胞源外泌体中的miRNA信号确实是导致肝癌恶性程度增加，包括增殖、侵袭迁移、EMT、索拉非尼耐药，的关键分子，其中miR1273f是促进EMT的核心信号，而细胞自噬是介导索拉非尼耐药的核心机制。