YB-1在肿瘤细胞来源的exosomes内稳定mRNA促进肿瘤血管新生的机制研究

Angiogenesis is an important factor for tumor progress。Tumor-derived exosomes can promote angiogenesis through inducing tumor-endothelial cell communication。In this procedure, mRNAs for VEGF and other related factors from tumor cells are important communicators, and horizontally transferred into endothelial cells to be translated into autocrine growth factors. Thus, it is necessary for mRNA to be kept stable during the transportation induced by exosomes. While the mechanism for the stability of mRNAs in exosomes is unclear. In cytoplasm, YB-1 combines with mRNAs and plays a key role in the stability of mRNAs, such as those for VEGF and other factors. Previous reports show the expression of YB-1 in the precursors of exosomes. For the first time, we manifested the existence of YB-1 in tumor-derived exosomes with in-vitro experiments. With clinical specimens, we found that strong YB-1 expression in tumor cells correlated with poor prognosis of patients and high microvascular density in tumor foci, and neovascular cells of tumor tissues also demonstrated high expression of YB-1. Therefore, our hypothesis is as follows: YB-1 involves in tumor-endothelial cell communication through combining and stabilizing mRNAs in exosomes, and thus promotes angiogenesis of tumor. The research will be done with cell clones with stably higher or lower expression of YB-1. The results will illustrate the mechanism of exosomes induced tumor-endothelial cell communication with more insights, and be helpful for the disclosure of new targets and the development of novel drugs for antiangiogenic therapy.

血管新生是影响肿瘤进展的重要因素。肿瘤细胞来源的exosomes可介导肿瘤细胞-内皮细胞的信息交流，促进肿瘤血管新生。VEGF等相关因子的mRNA作为信息载体，在exosomes内保持稳定，被运输至内皮细胞并翻译成自分泌因子,发挥关键作用。但是，mRNA在exosomes中保持稳定的机制不明。在细胞浆，YB-1是结合并稳定mRNA的核心因子，参与维持VEGF等因子的mRNA稳定。据报道YB-1存在于exosomes前体。我们在肿瘤细胞分泌的exosomes中也检测到YB-1；并发现肿瘤细胞YB-1表达与患者不良预后、癌灶微血管密度相关，YB-1还在肿瘤区新生血管高表达。故推测：YB-1参与肿瘤细胞-内皮细胞之间交流，在exosomes中结合并稳定VEGF等因子的mRNA，促进肿瘤血管新生。研究结果将进一步明确exosomes介导的肿瘤血管新生机制，为临床上寻找新的治疗靶点提供实验基础。

血管新生影响肿瘤进展。肿瘤细胞来源的外泌体（exosomes）可促进肿瘤血管新生。YB-1存在于细胞及exosomes前体中，可分别通过结合细胞内蛋白质以及稳定调节mRNA，影响肿瘤发生发展。本项目通过检测临床肿瘤标本的YB-1表达，肿瘤细胞内YB-1与肿瘤血管新生调节因子的相互作用，并采用肿瘤细胞分泌的exosomes作用于血管内皮细胞以模拟肿瘤微环境，探讨YB-1促进肿瘤血管新生的作用及相关机制。研究结果如下：1.163例外科手术胃癌组织的免疫组织化学分析显示YB-1表达与Lauren分型、JGCA分类、肿瘤血管浸润、微血管密度相关，YB-1在血管内皮细胞的表达与N、Ｍ、和TNM分期以及微血管密度相关，同时肿瘤血管与肿瘤细胞中的YB-1表达相关；另外，肿瘤细胞分泌的exosomes中检测到YB-1；提示YB-1在肿瘤细胞及肿瘤血管内皮细胞的表达均对肿瘤血管新生和肿瘤预后具有重要临床意义，同时YB-1可能在肿瘤细胞、血管内皮细胞以及介导二者对话的exosomes中均发挥调节肿瘤血管新生的作用。2.蛋白质免疫共沉淀、GST pull-down、和DNA松解实验等证实肿瘤细胞内YB-1直接结合并促进血管新生相关因子拓普异构酶1(TOPO1)的DNA松解作用，并受到血管新生相关因素氧化应激水平的调节。3. YB-1高表达胃癌细胞分泌的exosomes促进血管内皮细胞HUVEC增殖、侵袭、迁移和离体血管形成，而exosomes作用于HUVEC时发生内化，并导致HUVEC细胞发生上皮间质转化（EMT）和细胞内vimentin表达增加。4. YB-1高表达增加肿瘤细胞分泌的exosomes量及exosomes内YB-1水平， YB-1高表达细胞分泌的exosomes RNA行微阵列及DAVID分析显示，与血管新生相关因子的mRNA水平增加。进一步研究证实YB-1高表达细胞分泌的exosomes及其处理的HUVEC中的ang2、VEGF、VEGFR2、Tie2的mRNA水平增加。本课题的研究成果提示恶性肿瘤血管新生微环境中YB-1在肿瘤细胞中以及通过exosomes介导作用于血管内皮细胞而对肿瘤血管新生起着重要的作用。本研究结果将为进一步明确exosomes促肿瘤血管新生的作用，尤其YB-1的影响及其机制提供了有力证据，并为开发抗肿瘤血管新生的靶向治疗提供新思路。