vastatin/JAG2/Pck1信号通路在骨肉瘤转移中的抑制作用及其机制研究

Osteosarcoma (OS) has high incidence of metastases, especially lung metastasis, and the metastasis is difficult to be cured. Inhibition of angiogenesis is an important strategy for the treatment of metastasis. Our previous study found that type VIII collagen NC1 domain vastatin could inhibit vascular endothelial cells to form blood vessels, reduce the tumor bearing mice tumor microvessel density (MVD) and suppress metastasis, down-regulate the expression of JAG2 and Pck1, and do not change expression of vascular endothelial growth factor (VEGF). Meanwhile, we found that vastatin inhibited the invasiveness of OS cell line Saos-2. Specimens of OS patients with metastasis showed that vastatin expression was significantly decreased. In the contrast, the expression of JAG2 and Pck1 and tumor MVD was increased in specimens of OS patients with metastasis. Additionally, the expression level of VEGF had no obvious relation to the vastatin expression. The above results indicated that vastatin could inhibit blood vessel generation, thereby inhibiting osteosarcoma metastasis by JAG2/Pck1 signaling pathway independent of VEGF. This project intends to adopt pathway array to screen the important intermediate molecular between vastatin and JAG2 and Pck1. Furthermore, through Co-Ip assay, we will validate the molecular and sequence which specifically bind to vastatin, so as to clarify the molecular mechanisms of inhibition effect of vastatin to metastasis of OS. Then the large sample verification and functional recovery experiment will be administrated to find potential new therapeutic targets for metastasis of osteosarcoma.

骨肉瘤转移发生率高，疗效差。我们前期研究发现Ⅷ型胶原蛋白NC1结构域vastatin抑制血管内皮细胞形成血管，降低荷瘤鼠肿瘤微血管密度，抑制转移，先后下调JAG2和Pck1的表达，不影响VEGF表达；同时还能抑制骨肉瘤细胞的迁移侵袭。有转移的骨肉瘤患者标本中vastatin表达显著下降，并和JAG2和Pck1表达水平以及肿瘤微血管密度呈负相关，和VEGF表达水平则无明显相关性，提示vastatin可通过独立于VEGF外的JAG2/Pck1通路直接抑制骨肉瘤转移和抑制血管生成而间接抑制转移。本项目拟采用pathway array的实验方法，筛选vastatin 调控JAG2和Pck1的重要中间分子，并通过Co-Ip 等方法验证vastatin与这些分子之间的结合特异性及结合序列，阐明其在骨肉瘤转移中的分子机制，并通过大样本量验证、功能回复实验和PDX模型来寻找潜在的骨肉瘤肺转移新治疗靶点。

本项目前期研究发现vastatin基因能抑制血管内皮细胞形成血管，抑制转移，下调JAG2和Pck1的表达，并能抑制骨肉瘤细胞的迁移侵袭。通过进一步实验，首先进一步确认了vastatin可抑制MG-63骨肉瘤细胞的增殖，迁移，侵袭及裸鼠成瘤能力，此外vastatin还能抑制HUVEC的迁移、增殖和成管能力，进而抑制肿瘤微血管的形成。其次，通过大样本200例骨肉瘤组织标本的免疫组化，证实vastatin可抑制JAG2/Pck1的表达，且与肿瘤组织中肿瘤微血管密度的形成负相关，生存分析证实vastatin的表达预示骨肉瘤患者较好的预后。通过进一步生信分析及下游验证试验证实vastatin可能通过基因A发挥下游调控作用，之后的细胞学试验及生信分析发现基因A发挥抗肿瘤作用的机制并非涉及肿瘤细胞本身，而很可能通过血管内皮细胞（HUVEC）进而抑制肿瘤微血管生成来发挥作用，且这一作用很可能涉及JAG2/PCK1的调控。本项目的结论是，vastatin通过直接和间接双途径发挥抑癌作用：1.直接抑制肿瘤增殖，该机制通过JAG2/PCK1 的调控来发挥生物学作用；2.抑制肿瘤微血管生成，该作用通过调控基因A-JAG2/PCK1通路来发挥。鉴于肿瘤微血管形成在肿瘤的生物学机制中发挥重要作用，这一机制的进一步阐明对于研发针对肿瘤的个体化抗血管治疗具有很大的现实意义，这将通过后续相应验证试验来进行进一步的验证。