肿瘤相关成纤维细胞来源外泌体中miR-3164调控RAB5C促进骨肉瘤恶性进展的作用及机制研究

Osteosarcoma is the most common primary malignant bone tumor with poor prognosis and unclear pathogenesis. It has been shown that tumor associated fibroblast (TAF), key component of tumor microenvironment, plays a crucial role in the progression of osteosarcoma. Therefore, it’s of great importance to identify the exact communication mechanisms between TAFs and osteosarcoma cells. Based on our previous experiments and by comparing miRNA sequencing results of plasma exosome between 10 pairs of osteosarcoma patients and age-matched healthy donors, we found that TAF-derived exosomal miR-3164 can promote osteosarcoma progression. Further research reveals that miR-3164 elicits its pro-oncogenic function by targeting RAB5C, which is involved in the endocytosis, trafficking and recycling of E-cadherin. Hence, we suppose that exosomal transfer of TAF-derived miR-3164 promotes progression of osteosarcoma by targeting RAB5C. In this present study, we aim to clarify the exact mechanisms of TAF-derived exosomal miR-3164 in the development and progression of osteosarcoma, with CRISPR/cas9 techniques, conditional knockout mice, PDX models etc. In addition, we will determined whether plasma exosomal miR-3164 could serve as a feasible biomarker for the evaluation of therapeutic efficiency and monitoring of progression. The accomplishment of this study would provide a new strategy for the management of osteosarcoma.

骨肉瘤是最常见的原发恶性骨肿瘤，预后差且发病机制不清。研究显示在骨肉瘤进展中，肿瘤相关成纤维细胞（TAF）扮演重要角色，因而深入探索TAF与骨肉瘤细胞间相互沟通机制显得非常必要。课题组前期通过10对骨肉瘤患者与健康人血浆外泌体miRNA测序及实验发现：TAF分泌的miR-3164通过外泌体进入骨肉瘤细胞，增强其恶性潜能。进一步实验显示miR-3164直接调控RAB5C，而后者可能参与E-cadherin胞内循环转运。据此提出假说：TAF来源外泌体中miR-3164通过靶向RAB5C，干预E-cadherin循环转运而促进骨肉瘤恶性进展。本研究拟使用CRISPR/cas9技术、PDX模型、条件性基因敲除小鼠等进行多层面验证，阐明TAF通过外泌体中miR-3164促进骨肉瘤恶性进展的功能与机制；明确血浆外泌体miR-3164可否作为监测进展的液体活检指标，为改善骨肉瘤患者预后提供新的临床策略。

骨肉瘤是儿童与青少年最常见的原发性恶性骨肿瘤，极易发生肺转移。近十年来，骨肉瘤尤其是肺转移的治疗进入一个瓶颈期，无法取得明显的进展，亟待寻找新的研究方向及治疗靶点。大量研究表明肿瘤相关成纤维细胞TAF与肿瘤之间的细胞通讯十分活跃，二者共同促进了肿瘤的生物学进展。但骨肉瘤中肿瘤细胞与微环境TAF之间具体的通讯机制及相互作用尚不清楚。因此，本项目从骨肉瘤肿瘤微环境出发，首先建立骨肉瘤免疫分子分型，并分析发现TAF在分型中扮演重要角色。接着通过体内外实验明确TAF促进骨肉瘤肺转移的作用。通过10对骨肉瘤患者与健康人血浆外泌体miRNA测序及统计分析发现潜在功能分子miR-3164。进一步实验发现miR-3164来源于TAF分泌的外泌体，且可以靶向骨肉瘤细胞。体内外实验证实TAF来源外泌体中miR-3164靶向骨肉瘤细胞，增强其干性表型，促进肺转移形成。机制研究表明miR-3164通过靶向STK40激活p38-MAPK通路发挥作用。临床相关性分析显示TAF浸润与患者肺转移发生及新辅助化疗较差疗效相关。该项目研究成果为建立基于患者外周血外泌体miR-3164水平的疗效监测及肺转移预警模型提供理论依据。