骨肉瘤细胞源性外泌体lncRNA ENST00000506942.1介导转移前微环境调控在肺转移中的作用及机制研究

Osteosarcoma has a propensity to metastasize to the lung at early stage and with a high mortality rate. Recent studies suggested that exosoms played a critical role in the regulation pre-metastatic inche. Our previous research identified that the specific uptake of the osteosarcoma cell-derived exosomes occured in lung fibroblast cells and facilitated lung metastasis. Through sequencing and verification lncRNA ENST00000506942.1 was identified significant upregulated in exosomes. Furthermore knockdown this lncRNA in osteosarcoma cell influenced the downregulation of its level in exosomes. These exosomes were injected via tail vein, then low metastatic cell line ZOS were injected into these mice, and the number of lung metastatic node in the experiment group was substantially less than control group. Currently, the mechanisms and the role that are involved in the OS cell-derived exosome ENST00000506942.1 in lung pre-metastasis microenvironment have not yet been elucidated. Here,according to the previous studies, we plan to use osteosarcoma cell lines and animal models to verify the role of OS cell-derived exosome ENST00000506942.1 in lung pre-metastasis microenvironment; then identify the regulation network of ENST00000506942.1-micRNA-mRNA by means of RNA pulldown etc.; furthermore, according to clinical data, clinical significance of ENST00000506942.1 in exosomes would be analyzed. We aim to study the mechanisms and the role of OS cell-derived exosome ENST00000506942.1 in lung pre-metastasis microenvironment, with the purpose to shed light on the new therapeutic targets in lung metastasis.

骨肉瘤（OS）肺转移发生早、死亡率高。最新发现外泌体在转移前微环境调控中发挥重要作用。我们前期证实OS外泌体被肺成纤维细胞摄取并促进肺转移；高通量测序并验证发现外泌体lncRNA ENST00000506942.1表达显著上调；敲低该分子，外泌体中表达也下调；以敲低及对照组外泌体预处理动物，经尾静脉接种OS细胞，敲低组肺转移结节较少。目前OS外泌体ENST00000506942.1在肺转移前微环境中的作用及机制尚未阐明。现拟在前期基础上运用细胞、动物模型验证OS外泌体ENST00000506942.1在肺转移前微环境中作用；应用生物信息分析、RNA pulldown等鉴定lncRNA-miRNA-mRNA调控机制；结合病例资料，分析外泌体ENST00000506942.1临床意义。本项目旨在研究OS源外泌体ENST00000506942.1在肺转移前微环境中作用及机制，探索OS肺转移新靶标

骨肉瘤是青少年最常见的原发恶性骨肿瘤，其致死、致残率较高。随着新辅助化疗及外科手术技术的进步，目前骨肿瘤5年生存率提高到60%左右。研究表明80%骨肉瘤患者在临床诊断时已经存在肺部微转移灶，20%患者在确诊骨肉瘤时就已发现有临床确定的肺转移灶，其余在确诊时未发现肺部转移的患者20%~50%在治疗过程中发生肺转移。因此，控制骨肉瘤肺转移是进一步提高骨肉瘤患者生存率的关键所在。最新研究发现肿瘤细胞来源外泌体可通过介导与远处靶器官常驻细胞间的通讯，在靶器官转移前微环境调控中发挥重要作用。目前骨肉瘤细胞外泌体介导靶器官微环境调控的研究尚少。因此，本研究主要是探讨骨肉瘤细胞源性外泌体介导的靶器官微环境调控作用及机制，为骨肉瘤远处转移的治疗提供实验依据及理论指导。本研究我们发现骨肉瘤细胞来源外泌体主要是聚集在肺，并激活肺成纤维细胞，诱导肺转移前微环境的改变，促进肺转移的发生。通过对肺转移的患者血清外泌体进行高通量测序以及生物信息学，分析发现在之后后高转移的患者血清中lncRNA ENST00000506942.1存在显著高表达的情况。进一步研究，我们发现高转移株的骨肉瘤细胞分泌外泌体，并携带该lncRNA进入肺成纤维细胞，激活肺转移前微环境，促进骨肉瘤肺转移的发生。机制上，我们发现该lncRNA通过RBP结合的方式进入外泌体，促进肺体激活肺组织成纤维细胞炎性信号通路，从而诱导提高了骨肉瘤肺转移的发生率。应用生物信息学分析、基因转染、干扰、RNA pulldown等实验技术鉴定ENST00000506942.1的调控网络。最后，我们继续探究lncRNA ENST00000506942.1及其靶基因的临床意义研究，为寻找骨肉瘤治疗的新靶标提供理论依据及实验基础。