重楼总皂苷通过miR-520d-3p靶向调控Mig-7抑制骨肉瘤血管生成拟态形成及机制研究

Osteosarcoma, the most common bone sarcoma, causes a large amount of cancer-related death mainly due to fatal metastasis. Vasculogenic mimicry (VM) is a special type of vascular channel formed by tumor cells without endothelial cell participation. It plays an essential role in osteosarcoma metastasis due to its special structure which bypasses the barrier formed by endothelial cells and facilitates the leakage of tumor cells into the microcirculation. Therefore, identifying new drugs targeting VM formation offer a novel approach for osteosarcoma treatment. Our previous study (supported by NSFC General Fund No. 81673017) found that Rhizoma Paridis total saponins (RPTS), the major active component of Rhizoma Paridis possess anti-VM activity in osteosarcoma; however, its molecular mechanism remains unclear. Our previous data showed that Migration inducing gene-7 (Mig-7) is an important molecular target for osteosarcoma VM formation. RPTS decreased the expression of Mig-7. Then, what molecular signaling pathways get involved in the downregulation of Mig-7 after RPTS treatment? Bioinformatics software predicted Mig-7 to be miR-520d-3p targeting gene. Furthermore, Luciferase reporter gene assay and cellular transfection experiment confirmed that miR-520d-3p targeted Mig-7 for its downregulation. Moreover, qRT-PCR results demonstrated the upregulation of miR-520d-3p in osteosarcoma cells treated with RPTS. Therefore, we propose that RPTS inhibits VM in osteosarcoma through downregulation of Mig-7 targeted by miR-520d-3p. In this proposal, three-dimensional culture system, RNA interference, dual-luciferase reporter assay, immunofluorescent staining will be employed to elucidate the mechanism by which RPTS inhibit VM formation in osteosarcoma. We will also evaluate the anti-metastatic efficacy of RPTS in a bioluminescent orthotopic nude mice model of osteosarcoma by in vivo imaging. This proposal will not only provide scientific basis for anti-VM therapy in osteosarcoma, but also offer a new strategy for the drug development of Chinese herb medicine to inhibit tumor metastasis.

转移是导致骨肉瘤患者死亡的主要原因，血管生成拟态（VM）是骨肉瘤转移的重要因素。但治疗骨肉瘤VM药物仍相当缺乏。前期研究发现，重楼活性组分重楼总皂苷可抑制骨肉瘤VM，但分子机制不明。迁移诱导基因-7（Mig-7）是抑制骨肉瘤VM的重要靶点，前期研究证实，重楼总皂苷抑制骨肉瘤VM作用与调控Mig-7相关，那么重楼总皂苷如何调控Mig-7表达？软件预测和报告基因结果提示Mig-7是miR-520d-3p靶基因。此外，重楼总皂苷诱导骨肉瘤细胞miR-520d-3p显著上调。据此提出重楼总皂苷通过miR-520d-3p靶向调控Mig-7抑制骨肉瘤VM的科学假说。本项目拟应用三维培养、RNAi、荧光素酶报告基因检测、免疫荧光、裸鼠原位骨肉瘤模型、活体成像等技术研究重楼总皂苷抑制骨肉瘤VM和转移的作用及机制。本项目的实施为抗骨肉瘤VM治疗提供实验基础，并为抗肿瘤转移的中药新药开发提供新的思路。

骨肉瘤是青少年中最常见的原发性恶性骨肿瘤，转移是导致骨肉瘤治疗失败的主要原因，也是患者最终死亡的根本原因。血管生成拟态是骨肉瘤转移的重要因素。目前临床上仍缺乏有效的治疗血管拟态的方法及药物。前期研究发现，重楼活性组分重楼总皂苷可抑制骨肉瘤血管拟态，但分子机制不明。迁移诱导基因-7（Mig-7）是抑制骨肉瘤血管拟态的重要靶点，前期研究证实，重楼总皂苷抑制骨肉瘤血管拟态作用与调控Mig-7相关，那么重楼总皂苷如何调控Mig-7表达？软件预测和报告基因结果提示Mig-7是miR-520d-3p靶基因。此外，重楼总皂苷诱导骨肉瘤细胞miR-520d-3p显著上调。由此提出重楼总皂苷通过miR-520d-3p靶向调控Mig-7抑制骨肉瘤VM的科学假说。本项目采用应用体外三维培养、荧光素酶报告基因系统、细胞转染、鬼笔环肽染色、构建胫骨原位骨肉瘤裸鼠模型等技术在细胞、分子及整体水平上研究重楼总皂苷抑制骨肉瘤血管拟态和转移的治疗效果及作用机制。研究结果发现重楼总皂苷可以显著抑制多种骨肉瘤细胞迁移运动和拟态形成，其机制与下调Mig-7和上调miR-520d-3p有关。在6种骨肉瘤细胞和人成骨细胞中miR-520d-3p与Mig-7蛋白表达呈负相关，Mig-7是miR-520d-3p的靶基因。miR-520d-3p/Mig-7信号轴通过调控下游PI3K/AKT/mTOR/MMPs/Ln-5γ2信号通路和细胞伪足的形成，重塑胞外基质和重构细胞骨架，影响了骨肉瘤血管生成拟态和骨肉瘤原位裸鼠模型肺转移灶的形成。在以上研究基础上，我们进一步采用miR-520d-3p抑制剂或Mig-7表达质粒转染骨肉瘤细胞，证实重楼总皂苷显著抑制胞外基质重塑和细胞骨架重构，从而发挥抑制骨肉瘤血管拟态形成及骨肉瘤转移的药效具有miR-520d-3p/Mig-7信号轴依赖性。本项目的实施为抗骨肉瘤血管拟态治疗提供实验基础，并为抗肿瘤转移的中药新药开发提供新的思路。本研究以第一作者发表SCI论文4篇，完成预定目标。