胃癌细胞来源外泌体miR-519a-3p通过抑制肝Kupffer细胞Lefty1分泌以促进肝转移灶形成的机制研究

The liver metastasis of gastric cancer is one of the key factors that affect the prognosis of patients. Exosome has been proved to be involved in the process of metastasis of various tumors. Our workgroup found that exosomes derived from gastric cancer cells were mainly absorbed by liver Kupffer cells (KCs). High throughput sequencing suggested that exosomal miR-519a-3p derived from gastric cancer cells was associated with liver metastasis. In vivo experiments confirmed that it had a promoting effect on liver metastasis. The mRNA microarray analysis and functional experiments revealed that exosomal miR-519a-3p could inhibit the expression and secretion of Lefty1, a stemness-associated factor in KCs. In addition, mutating AGAG fragment in the sequence of miR-519a-3p could reduce its expression in exosomes. Accordingly, we put forward the hypothesis that the exosomal miR-519a-3p derived from gastric cancer cells is absorbed by KCs, inhibiting the expression of Lefty1, enhancing the stemness of disseminated gastric cancer cells in liver and accelerating the process of liver metastasis. The AGAG fragment is associated with the process of assembling miRNA into the exosomes. We will employ cell lines, organoids, clinical samples and animal models to validate the hypothesis and provide the scientific evidence for the precision therapy of liver metastasis of gastric cancer via exosome tracing, RNA pull-down and other modern techniques.

胃癌肝转移是影响患者预后的关键因素之一，外泌体已被证实参与多种肿瘤的转移过程。本课题组前期研究发现胃癌细胞来源外泌体主要被肝Kupffer细胞（KCs）吸收，高通量测序提示胃癌细胞来源外泌体miR-519a-3p与胃癌肝转移相关，体内实验表明其可促进胃癌肝转移灶形成；mRNA芯片分析及功能实验揭示外泌体miR-519a-3p可抑制肝KCs内干性相关因子Lefty1的表达及分泌。此外，突变miR-519a-3p序列中AGAG片段可降低其在外泌体中的表达。据此我们提出假说：胃癌细胞内miR-519a-3p受其序列上AGAG片段调控进入外泌体并被肝KCs吸收，抑制Lefty1表达及分泌，增强定植在肝内胃癌细胞干性，促进肝转移灶形成。本研究拟从细胞系、类器官、临床样本及动物模型四个层面，通过外泌体示踪、RNA pull-down等技术对假说进行证实，为胃癌肝转移的精准化治疗提供科学依据。

胃癌发生肝转移是影响患者预后的关键因素之一，且肝脏又是胃癌血运转移的主要靶器官。外泌体已被证实参与了多种肿瘤的靶向转移过程；然而，胃癌来源外泌体及其内容物在胃癌肝转移过程中究竟起到何种作用，其中机制为何仍有待进一步研究。本项目针对上述科学问题，围绕揭示胃癌细胞外泌体对胃癌肝转移的作用及其主要的吸收器官和细胞、明确影响胃癌肝转移的外泌体miRNA靶标、阐明胃癌外泌体内具体miRNA靶标对胃癌肝转移的作用及机制、揭露具体miRNA靶标进入外泌体的上游调控机制四个研究目标开展工作，取得了以下重要研究结果：本项目建立了胃癌外泌体训导的裸鼠胃癌肝转移模型及高度肝转移潜能的胃癌细胞株，并利用上述模型发现高度肝转移潜能胃癌细胞来源的外泌体可促进胃癌肝转移进程。组织活体成像、免疫荧光等实验结果表明，胃癌细胞来源外泌体主要被肝脏吸收，且主要的效应细胞为肝巨噬细胞。基于小鼠模型及临床样本的相关实验发现胃癌细胞来源外泌体miR-519a-3p对胃癌肝转移具有促进作用，且其表达量与胃癌肝转移患者预后呈显著负相关。分子机制方面，利用荧光素酶报告基因、表达量相关性检测、流式细胞学等一系列体内外实验，我们证明了胃癌外泌体miR-519a-3p可直接靶向肝巨噬细胞内的ERK去磷酸化酶DUSP2并抑制其表达，从而激活MAPK/ERK信号通路并诱导巨噬细胞M2极化。ELISA、小鼠主动脉环出芽、人静脉血管生成等实验进一步揭示了胃癌外泌体miR-519a-3p可通过调控DUSP2-MAPK/ERK轴促进巨噬细胞TGF-β、VEGF-A和VEGF-D分泌及肝微环境内血管形成。最后，利用生物信息学分析、RNA pull-down实验、基因编辑技术及临床样本验证，本项目还发现胃癌细胞内的RNA结合蛋白hnRNPL可结合miR-519a-3p成熟序列上的AGAG片段并指导其进入外泌体。综上所述，上述关键研究结果补充完善了现有关于胃癌肝转移分子机制的理论背景；在后续临床研究的验证下，胃癌来源外泌体miR-519a-3p有望成为胃癌肝转移的新型诊断标志物及治疗靶点。