COPS3促进骨肉瘤肺转移的体内实验及其相互作用蛋白的鉴定

Osteosarcoma (OS) is the most common primary malignant tumor of the bone. It is characterized by a local aggressiveness, with a tendency to metastasize to the lung in children and young adolescents. Despite of multimodal therapy over the past 35 years, pulmonary metastasis remains the "bottleneck" of further improving survival of patients with OS. The COPS3 gene was recently identified to be an oncogene, which amplified and overexpressed in osteosarcoma. After several years of research, we first reported that the overexpression of the COPS3 protein is significantly correlated with pulmonary metastasis in OS patients (p<0.05). Inhibiting COPS3 function by siRNA in vitro resulted in reduced osteosarcoma cell proliferation and migration (p<0.01), and MAPK signaling pathway was also downregulated at the same time. Encouraged by the above promising results, we determined to continue our COPS3 gene research. Firstly, we will explore the expression pattern of COPS3 protein in paired OS　specimens from biopsy before chemotherapy and resected lung metastasis lesion. Secondly, we aim to undergo in vivo study to attest the reduction of lung metastases by COPS3-SiRNA. Finally, co-immunoprecipitation, mass spectrometry and western blot will be applied to screen and identify interacting proteins of COPS3. Some COPS3 target proteins will be selected and their functions analyzed. The mapping of the complex network of protein-protein interaction might lay a foundation for further understanding in its signal transduction pathway. We believe that this project will offer more evidence of the lung metastasis promotion by COPS3 and facilitate in finding novel targets to treat pulmonary metastasis in the near future.

骨肉瘤是儿童和青少年最常见的高度恶性原发骨肿瘤，肺转移是患者死亡的主要原因，因此肺转移是目前临床上限制骨肉瘤患者生存率进一步提高的"瓶颈"。COPS3是新近发现的在骨肉瘤组织中有扩增和过量表达的癌基因。申请者多年的系列研究首次证实了该基因扩增和蛋白高表达与骨肉瘤肺转移显著相关（p<0.05）；体外敲除COPS3基因，骨肉瘤细胞的迁移和侵袭能力显著降低（p<0.01）。本课题是延伸性研究，观察肺转移标本中COPS3的表达；动物实验观察COPS3基因敲除后对肺转移的影响；运用免疫共沉淀，质谱分析和Western blot筛选和鉴定与COPS3相互作用的目的蛋白。通过对目的蛋白的功能注释和相互作用网络构建，为进一步明确其信号传导机制奠定基础。本课题的研究将进一步证实COPS3对骨肉瘤肺转移的促进作用，并通过相互作用蛋白的鉴定，对其信号传导途径和发现在临床上治疗肺转移的可能的靶点起到一定积极作用。

肺转移是骨肉瘤患者死亡的主要原因，而目前临床上对骨肉瘤肺转移缺乏有效的早期发现和治疗手段。我们前期研究发现COPS3的扩增与过表达与骨肉瘤肺转移显著相关。本研究对比配对原发灶与肺转移标本中COPS3的表达，体内实验干扰COPS3表达对肺转移的影响，并运用免疫共沉淀筛选和鉴定COPS3的相互作用蛋白，进一步探究其信号传导途径。研究发现相对于原发灶，肺转移灶中COPS3表达进一步提高；小鼠体内实验证明干扰COPS3表达显著抑制骨肉瘤肺转移；免疫共沉淀证实COPS3与Raf-1存在相互作用，干扰COPS3使Raf-1表达降低并抑制其下游MEK/ERK/RSK通路的活性，相反过表达COPS3使Raf-1表达增加并激活其下游通路，且骨肉瘤细胞转移能力与该通路活性变化相一致。这些结果共同表明COPS3通过与Raf-1相互作用激活了MEK/ERK/RSK通路从而促进了骨肉瘤肺转移。以COPS3为靶点的治疗可能会成为临床上一项治疗骨肉瘤转移的有效措施。