PlGF调节血管拟态形成在骨肉瘤耐药中的作用机制

Osteosarcoma is the most common primary solid tumor of bone in children and young adults, comprising about 20% of primary bone sarcomas. Currently, the primary treatments of this disease include chemotherapy and surgery. However, most chemotherapeutics carry the risk of both short-term and long-term toxic effects.The emerging knowledge of the pathogenesis and genetic abnormalities associated with osteosarcoma has exposed the field to new potential molecular targets. Tumor growth is dependent on the degree of neovascularization in the tumor bed. Vasculogenic mimicry (VM), a formation of nonendothelial microvascular channels, has been generally recognized as a newpattern of neovascularization in aggressive malignancies. Several authors have reported a process termed vasculogenic mimicry (VM), which consists of the formation of patterned, tubular networks by aggressive tumor cells, that mimics endothelial-formed vasculogenic networks. VM refers to the unique ability of highly aggressive human tumor cells and the channel formed by VM is composed of a basement membrane and is lined by tumor cells. VM patterns serve as an adjunct to the existing vasculature system, thereby aiding tumor growth and contributing to the metastatic process. Cells capable of VM display a high degree of plasticity, causing them to resemble dedifferentiated cell types. VM is characterized by an undifferentiated molecular signature together with embryonic-like differentiation plasticity, thereby implying a link between CSCs and aggressive tumour cells capable of VM. Indeed, these two cell types share the potential for unlimited proliferation potential, cellular plasticity and the expression of genes involved in maintaining pluripotency and/or plasticity. Previous studies showed that high indexes of VEGF expression were significantly associated with osteosarcoma recurrence, drug resistance and death. PlGF shares 42% amino acid sequence identity with VEGF and appears to be crucial for pathological angiogenesis，apoptosis of tumor cell and drug resistance. Some studies have demonstrated that PlGF collerate with angiogenesis, apoptosis of tumor cell and the multidrug resistance of tumor. The Notch signal parthway can induce angiogenesis of tumor and correlate with proliferation of tumor cell. Our previous study found that the expression of PlGF and Notch1 were higher in U2-OS cells and were significantly correlated with tumor diameter and Enneking staging but were independent with tumor site、age and sexuality of patient. The proliferation of osteosarcoma cell were inhibited when we downregulated the expression of PlGF gene. But the definite mechanisms were unkown. So we suppose that PlGF may induce the proliferation and drug resistance of osteosarcoma cell through Notch signal pathway and formation of VM patterns. In order to validate the effect of PlGF on multidrug resistance of osteosarcoma, we will induce U2-OS cell into multidrug resistant cell, regulate the expression of PlGF and Notch signal pathway, then evaluate cell proliferation and chemotherapy sensibility, and explore its influence on the apoptosis, proliferation,and drug resistance of osteosarcoma in vitro and in vivo by regulation the gene expression of PlGF and Notch1 with the RNA interference technique or gene transfection. Accordingly, we can explore the effect and mechanism of PlGF on drug resistance in osteosarcoma. Hence, this work may provide an new insight into oncotherapy and has favourable perspective in the clinical treatment of osteosarcoma.

骨肉瘤细胞内皮化后具备肿瘤干细胞特性，形成血管拟态（VM)，参与肿瘤耐药。VEGF可调节内皮细胞形成新生血管，但不能阻止VM形成，因此VM被认为可导致治疗失败和肿瘤恶性进展。研究发现胎盘生长因子(PlGF)与肿瘤耐药相关，PlGF可逆转Notch抑制剂诱导的非正常肿瘤血管变成功能化的微血管网络，提示其在VM形成中的作用，但具体分子机制尚不明确。我们前期研究发现PlGF和Notch1在骨肉瘤耐药细胞中高表达，下调PlGF表达后，细胞增殖明显抑制。因此我们推测PlGF在骨肉瘤中可能通过Notch1信号通路，诱导肿瘤微环境中血管拟态形成，促进肿瘤细胞增殖和侵袭，导致耐药发生。为了验证此假设，本研究拟在耐药骨肉瘤细胞中调控PlGF和Notch1表达，检测Notch1靶基因、耐药转运蛋白和VM等微环境改变，明确调控PlGF在逆转骨肉瘤耐药中的作用及机制，本课题将为骨肉瘤的综合治疗提供有益的实验依据。

肿瘤多药耐药是疗效不佳的主要因素，胎盘生长因子(PlGF)属VEGF家族，与肿瘤细胞凋亡、耐药和血管生成相关，Notch信号通路可增强VEGF的作用，参与骨肉瘤的增殖和凋亡。我们前期研究发现PlGF和Notch1在骨肉瘤高表达，且与肿瘤临床分期相关。下调骨肉瘤细胞PlGF表达后，细胞增殖明显受抑制，但PlGF抑制骨肉瘤增殖的具体分子机制尚不明确。我们推测PlGF在骨肉瘤中通过影响Notch信号通路，促进增殖，诱导耐药，可能是骨肉瘤治疗效果不佳的分子靶点之一。本研究诱导建立肉瘤耐药细胞，干预PlGF的表达，评估细胞增殖和化疗敏感性，检测细胞增殖、耐药基因和Notch信号通路因子表达，研究PlGF在细胞增殖和化疗敏感性中的作用。研究结果显示通过RNA干扰技术调控骨肉瘤耐药MG-63细胞中PlGF基因的表达，可以显著降低实验组中PlGF蛋白的表达，同时在转染后的不同时间点肿瘤细胞对化疗药物的敏感性明显增加，肿瘤细胞凋亡较空白对照组和阴性对照组显著增加，本研究认为PlGF可作为逆转骨肉瘤耐药的基因干预靶点，干预其表达可显著增加骨肉瘤耐药的化疗敏感性。本研究将为骨肉瘤综合治疗提供必要的实验依据。