Exosomal miRNA-21-5p通过骨髓前体细胞诱导结直肠癌细胞MET促进肝转移的机制研究

Clinically, colorectal carcinoma (CRC) metastasis to the liver is the main reason of CRC-related death. Mesenchymal-epithelial transition (MET) has been suggested to be essential for the colonization of metastatic carcinoma cells in distant organs or tissues. To date, however, the underling mechanisms by which drive carcinoma cells MET, as well as their colonization in metastatic lesions, remain largely unclear. In a previous study from our group, we built a six miRNAs-based classifier to efficiently predict the metastasis and/or prognosis of CRC patients in stage II (Lancet Oncology, 2013 Dec;14(13):1295-306). Recently, we identified that miR-21-5p, one member among the six miRNAs classifier, was predominantly existed in CRC cellular exosome, and high levels of exosomal miR-21-5p, either in patients peripheral blood or tumor tissues, were positively correlated with CRCs advanced clinical stages and/or liver metastasis. In an in vivo metastasis model of mouse, we further observed that exosomal miR-21-5p extracted from CRC cells culture medium could substantially promote CRC cell metastasis in the liver and induce MET. Furthermore, in our in vitro and in vivo studies, in which a constructed bone marrow-reconstituted mouse mode was used, our data strongly suggested that exosomal miR-21-5p is able to drive CRC cells MET and support their colonization and metastases to the liver via enhancing the bone marrow progenitor cell (BMPC)s mobilization and their final recruitment to the liver. To further investigate and discover the functions and/or mechanisms of exosomal miR-21-5p in promoting CRC cells MET and metastasis in the liver, in the present project, we designed a series of additional in vivo to in vitro experiments. The purpose of our project is 1) to verify the subgroups of BMPCs regulated and recruited in the liver by CRC cells exosomal miR-21-5p, 2) to explore the underling mechanisms of exosomal miR-21-5p-induced BMPCs mobilization and colonization in the liver, 3) to identify the functions and/or mechanisms of the subgroups of BMPCs, including their secreted key cytokines, which may regulate CRC cells MET in the liver, and 4) to discover the clinical significances, as well as the correlations between the levels of exosomal miR-21-5p, the subgroups of BMPCs and their key cytokines which was identified to regulate CRC cell MET, in peripheral blood from a large case of CRC patients in different clinical stages. In summary, by performing this project, we will do our hard to elucidate the underling mechanisms by which exosomal miR-21-5p promotes CRC cells MET and liver metastases through regulating specific BMPC subgroups move to the liver. We trust that the data from our present project will provide more experiment evidences for uncovering the precise mechanisms of CRC liver metastasis, so as to find novel therapeutic targets and develop new modalities of treatment.

结直肠癌（CRC）肝转移是病人死亡的主要原因。癌细胞间质-上皮转化（MET）是其定植于转移灶的重要条件, 但机理不清楚。我们前期发现miR-21-5p等6联miRNAs可有效预测II期CRC转移；最近我们建立了骨髓重组小鼠模型，发现CRC细胞外泌体miR-21-5p可募集骨髓前体细胞（BMPCs）至肝脏并促进癌细胞MET和肝转移。本项目拟承前启后，将通过一系列体内外实验，明确外泌体miR-21-5p调控的BMPCs亚群；解析miR-21-5p动员BMPCs的分子机理；探究募集至肝的BMPCs促进CRC细胞MET等功能与机制；确证CRC病人外周血中miR-21-5p、BMPCs亚群及其调控MET关键因子水平与肿瘤肝转移的相关性及临床价值；深入阐明外泌体miR-21-5p通过募集BMPCs促进CRC细胞MET和肝转移的确切机制和肿瘤学意义，为揭示CRC肝转移机理和抗癌治疗新靶点提供实验研究基础

结直肠癌（CRC）肝转移是病人死亡的主要原因。癌细胞间质-上皮转化（MET）是其定植 于转移灶的重要条件, 但机理不清楚。我们前期发现miR-21-5p等6联miRNAs可有效预测II期C RC转移；我们通过骨髓重组小鼠模型，发现CRC细胞外泌体miR-21-5p可募集骨髓前体细 胞（BMPCs）至肝脏并促进癌细胞MET和肝转移。本项目致力于研究结直肠癌（CRC）癌细胞通过外泌体来源的miR-21-5p通过募集骨髓前体细胞诱导CRC细胞间质-上皮转化（MET）定植于转移灶的机制。通过临床标本检测及一系列体内外功能实验发现：miR-21 -5p在CRC组织中高表达，且其表达水平与CRC患者淋巴结转移及远处脏器转移明显正相关；证实Exosomal miR-21-5p可直接促进基因重组小鼠骨髓前体细胞动员能力；骨髓重组小鼠原位转移模型的肝脏中分选出的骨髓细胞亚群条件培养基能够增强CT26的增殖和生长能力，降低其迁移能力；通过抗体芯片检测发现，经过表达miR-21-5p组外泌体处理后GFP+PKH26+ 群细胞分泌的多个细胞因子的量有明显增强，发现骨髓来源细胞分泌的X, Y及LIF因子具有促进肠癌细胞MET的作用；而干扰小鼠肠癌细胞CT26中其细胞因子的受体可部分阻断其诱导MET的作用。目前相关细胞因子的中和抗体正在优化和专利申请中。本项目为揭示CRC肝转移机理和抗癌治疗新靶点提供实验研究基础，研发的中和抗体具有潜在转化价值。