骨肉瘤血管生成拟态靶向治疗的新分子靶点初探

Osteosarcoma, the most common bone sarcoma, causes a large amount of cancer-related death in children and young adults mainly due to development of fatal metastasis, usually in the lungs. There are multiple mechanisms by which osteosarcoma can develop and maintain their vascular supply. These mechanisms allow tumors to continue to grow in size, invasion and metastasize. Vasculogenic mimicry(VM) is a new and special microcirculation pattern independent of conventional angiogenesis and vasculogenesis, in which highly aggressive tumor cells mimic endothelial cells and form vascular channel-like structures to convey blood plasma and red blood cells without the participation of endothelial cells. VM is more inclined to facilitate the transimission of tumor cells in the process of metastasis aside from supplying blood and nutrients. Therefore, VM is also associated with a poor prognosis, worse survival and the highest risk of cancer recurrence for patients with malignant tumors. One of the tumor cell proteins that facilitate their invasion, Migration inducting gene-7 (Mig-7), has been discovered in recent years. Expression of Mig-7 is limited to tumor cells and to date not found in normal tissues. Quite a lot of data strongly suggest that Mig-7 plays an important role in the processes of VM. In our recently studies, Mig-7 mRNA and protein expression were detected in 143B and MG63 human osteosarcoma cell lines for the first time, and VM was found in 15 of the 66 osteosarcoma samples (22.7%). On the basis of our previous work, we are going to investigate the following aspects in this research. . The expression of Mig-7 in operation specimen and blood serum of osteosarcoma will be examined. The relationship between Mig-7 expression and the osteosarcoma progression and metastasis will be analyzed using large sample data. The effect that alteration to Mig-7 expression in osteosarcoma cells by gene transfection and RNA interference would have on the capability of invasion, cell polarity and vessel-like structure formation in three-dimensional cultures will be investigated. Based on CD34 and PAS double staining, the VM structures will be observed after lentivirus-mediated RNAi knockdown or overexpression of Mig-7 in osteosarcoma orthotopic transplantation animal model. The prognosis of the animal will also be recorded.. On these grounds, we are going to explore the role of Mig-7 in VM progression of osteosarcoma and related mechanism in order to look for a potential candidate for future targeted anti-vascular tumor therapies.

血管生成拟态(VM)是一种特殊的肿瘤微循环结构，比内皮依赖性血管更易出现血行转移，其治疗尚无有效靶点。迁移诱导基因-7(Mig-7)在多种恶性肿瘤的VM形成中发挥重要作用，却不在正常组织中表达。骨肉瘤血供丰富，预后差。我们发现人骨肉瘤细胞Mig-7呈阳性表达，并在骨肉瘤组织中观察到VM结构，但Mig-7对骨肉瘤VM形成及预后转归的影响至今未见报道，其机制更不明晰。.因此，本课题拟在前期研究基础上考察：1、检测大样本患者骨肉瘤组织和血中Mig-7的表达并长期随访，分析其与骨肉瘤VM形成及预后的相关性；2、通过基因转染和RNA干扰技术体外分析Mig-7表达改变对骨肉瘤细胞侵袭、细胞极性以及形成拟态样三维管腔结构的影响；3、构建慢病毒载体，特异性增强或阻抑Mig-7表达，观察骨肉瘤动物模型中VM及预后转归的变化。.据此，探讨Mig-7在骨肉瘤VM中的作用及机制，寻找抗血供靶向治疗的新靶点。

血管生成拟态（Vasculogenic mimicry, VM）是一种有别于传统肿瘤血管生成的微循环模式，它不依赖于机体血管内皮细胞。而迁移诱导基因-7（Migration-inducing gene 7, Mig-7）在多种恶性肿瘤的VM活动中发挥重要作用。前期研究中我们发现骨肉瘤中存在VM现象和Mig-7蛋白表达。本研究主要目的是揭示Mig-7对骨肉瘤VM的影响及可能的机制。本项目在四肢原发性骨肉瘤患者的组织学观察和随访中证实，Mig-7染色主要位于骨肉瘤细胞的胞浆和胞膜，VM及其相关分子Mig-7均与病情进展密切相关，两者均为骨肉瘤的独立预后因素。随后我们建立了骨肉瘤细胞体外三维培养体系，并发现敲除Mig-7可以显著抑制骨肉瘤细胞在体外生成拟态样结构的能力，且研究显示Mig-7通过影响细胞的运动和侵袭能力而非生存增殖速度来调控骨肉瘤VM形成。进一步，我们通过基因芯片技术提供了与高运动侵袭性143B骨肉瘤细胞株VM表型相关的lncRNAs和mRNAs表达谱；构建了lncRNA-mRNA共表达网络图并识别出关联度最高的lncRNAs是n340532；绘制基因作用关系网络图寻找核心基因，并选择处于关键节点位置的ITGA2用于后续研究。初步验证显示，敲低n340532和ITGA2都可以抑制体内外骨肉瘤VM形成和骨肉瘤在体内的生长及肺转移。最后，我们将表达Mig-7 shRNA的重组慢病毒载体转染骨肉瘤细胞并植入裸鼠胫骨髓腔，建立荷瘤动物模型。在体研究显示，抑制Mig-7表达能够减少骨肉瘤病灶中VM结构的形成，迟滞肺转移并延长动物的生存时间。综上，本研究提示Mig-7通过改变肿瘤细胞的侵袭能力促进了骨肉瘤VM形成，并由此促进病程进展，抑制Mig-7表达则可以阻抑这一过程。通过本项目的研究，丰富了骨肉瘤VM形成及相关机制的理论，并为骨肉瘤的抗VM治疗提供了新的潜在靶点。