前列腺肿瘤外泌体特异传播miR-378促进成骨前体细胞分化及骨转移的机制研究

The high mortality of prostate cancer is mainly caused by its bone metastasis, of which the molecular mechanism remains unclear. It has been reported that tumor cells can interact with recipient cells in microenvironment by exosomal microRNA to promote tumor metastasis. Our previous studies identified an elevated expression of miR-378 in the serum exosomes in patients with bone metastatic prostate cancer. In addition, miR-378 is highly enriched in exosomes derived from prostatic orthotopic xenograft in nude mice by orthotopic inoculation of human prostate cancer cell line LNCaP cells. After incubation murine pre-osteoblast cell line MC3T3-E1 cells with miR-378 enriched exosomes, the expression of Gli3 (a repressive component of SHH pathway) and Schnurri3 (an inhibitory factor of pre-osteoblast differentiation) is downregulated, and the expression of pre-osteoblast differentiation markers is upregulated. Based on these results, in this project we attempt to investigate whether and how tumor derived exosomal miR-378 promotes pre-osteoblast differentiation and bone metastasis of prostate cancer via regulation of SHH and other signaling pathway as a mechanism, using stable miR-378 overexpression or miR-378 knockdown prostate cancer subclone cell lines, nude mice orthotopic and intertibial xenograft models and knockout mouse models. By elucidation of relevant mechanism, our findings may provide novel clues and targets for the therapy of prostate cancer with bone metastasis.

前列腺癌的骨转移是其高致死率的主要原因，但相关的分子机制仍不明了。已有的研究表明，肿瘤细胞通过分泌外泌体传播microRNA实现与微环境中受体细胞的相互作用，促进肿瘤转移。我们的前期研究发现，在前列腺癌骨转移病人血清外泌体中miR-378呈高表达。此外，miR-378在前列腺癌细胞系LNCaP裸鼠前列腺原位植瘤组织来源的外泌体中呈高表达。小鼠成骨前体细胞系MC3T3-E1细胞与富含miR-378的外泌体孵育后，Gli3（SHH通路的抑制型组分）与Schnurri3（成骨前体细胞分化抑制因子）表达下调，成骨前体细胞分化标志物的表达上调。本项目在上述研究基础上运用过表达或稳定敲降miR-378的前列腺癌细胞亚克隆系、裸小鼠植瘤及基因敲除小鼠模型，以miR-378为切入点探讨其如何通过调控SHH及其他信号通路促进成骨前体细胞分化及前列腺癌骨转移的机制，为治疗前列腺癌骨转移提供新的线索和靶标。

项目背景：.前列腺癌的骨转移是其高致死率的主要原因，但相关的分子机制仍不明了。我们的前期研究发现，在前列腺癌骨转移病人血清外泌体中miR-378呈高表达。此外，成骨前体细胞（BMSC）与富含miR-378的肿瘤外泌体孵育后，BMSC分化标志物的表达上调。本项目运用相关前列腺癌细胞亚克隆系及对应小鼠模型，以miR-378为切入点探讨其如何调控相关信号通路构建促肿瘤转移微环境并促进前列腺癌骨转移的分子机制，为治疗前列腺癌骨转移提供新的线索和靶标。.主要研究内容：.1）探讨在前列腺癌细胞中过表达miR-378 并削弱外泌体的释放能否及如何抑制肿瘤细胞的生长/转移。.2）验证前列腺癌外泌体传播miR-378 是否及如何促进成骨/溶骨前体细胞分化建立相应微环境。.3）验证分化成熟的成骨/溶骨细胞是否及如何通过分泌相关的细胞因子和（或）生长因子促进前列腺癌细胞在转移部位的生长。.研究结果：.1）前列腺癌细胞中过表达miR-378并削弱其经由外泌体的释放抑制肿瘤生长转移。.2）在体外2D或3D培养时过表达miR-378均可促进BMSC的分化。.3）在体内发现富含miR-378外泌体可同时被溶骨前体细胞（BMM）吸收诱导溶骨分化促进前列腺癌骨转移。.4）分化成熟的溶骨细胞分泌ANGPTL2促进前列腺癌细胞增殖。.关键数据及科学意义：.1）维持前列腺癌细胞中高表达miR-378靶向MAOA抑制EMT发生以此抑制肿瘤的生长转移。.2）过表达miR-378后在体外2D培养5天即可显著提高成骨关键基因RUNX2及标志物OB-Cad的表达。.3）前列腺癌中hnRNPA2B1高表达介导了miR-378在外泌体中的富集。.4）miR-378靶向Dyrk1a促进溶骨分化并增强ANGPTL2表达分泌。.5）本项目研究成果提示了阻断miR-378向BMSC及BMM传递对于抑制肿瘤骨转移具有积极的临床意义。