代谢型谷氨酸受体4在人骨肉瘤发生发展中的作用机制研究

Osteosarcoma is the most common primary malignant bone tumor. The foreigner made genome-wide association analysis study and found that the rs1906953 of mGluR4 gene is related to osteosarcoma; also, our previous research has found that the expression of mGluR4 in osteosarcoma is significantly higher than that of the normal bone tissue, and patients with osteosarcoma of high mGluR4 expression has poor prognosis. On this basis, to explore the functions of mGluR4 in the occurence and development of osteosarcoma, the methylation-specific PCR method will be used to make methylation inspection to the promoters;the method of PCR allele classification will be used to test for the polymorphism of mGluR4 gene in both patients with osteosarcoma and in normal people;after screening out the osteosarcoma cell line with high expression of mGluR4, we respectively use the agonist and inhibitor of mGluR4, and use the established RNAi lentiviral expression vector to treat the osteosarcoma cells, investigate the changes on the cell cycle, proliferative activity, invasion ability, tumor formation capacity in nude mice and other biological behaviors; finally, we will use real-time fluorescent quantitative PCR array analysis technology to explore the signaling pathways and targets of the influence of mGluR4 gene on osteosarcoma,we propose to use Western Blot、Immunofluorescence and Co-Immunoprecipitation techniques to identify it,and to explore the mechanism of the occurrence and development of osteosarcoma from molecular biology.

骨肉瘤是最常见的原发恶性骨肿瘤。国外的全基因组关联分析发现，mGluR4基因的rs1906953与骨肉瘤相关，我们的前期研究也发现，mGluR4在骨肉瘤中的表达高于正常骨组织，且具有mGluR4高表达骨肉瘤患者的预后较差。我们拟采用甲基化特异性的PCR法，对mGluR4基因进行启动子甲基化检测；采用PCR等位基因分型检测mGluR4基因在骨肉瘤患者和正常人群中的多态性；筛选出具有mGluR4高表达的骨肉瘤细胞系后，分别采用mGluR4的激动剂、抑制剂以及构建的RNAi慢病毒表达载体，对骨肉瘤细胞进行处理，观察细胞周期、增殖活性、侵袭能力和裸鼠成瘤能力等生物学行为的变化情况；最后，我们还将采用实时荧光定量PCR阵列分析技术探索mGluR4基因对骨肉瘤产生影响的信号通路和作用靶点，并利用免疫蛋白印迹、免疫荧光染色及免疫共沉淀等技术进行验证，从分子生物学上探讨人骨肉瘤发生、发展的作用机制。

骨肉瘤是最常见的原发恶性骨肿瘤，以恶性程度高和预后差为主要特征，是儿童和青少年癌症相关的主要死亡原因。它的发病机制仍是不清楚的。因此，我们需要对骨肉瘤的发病风险和预后标记进行更深入的研究，从而寻找新的治疗靶点。.在本项目的研究中，我们研究发现：在中国人群中mGluR4基因rs19066853单核昔酸多态性与骨肉瘤的易感性有关，可能会增加患骨肉瘤的危险。携带rs19066853 CC基因型的骨肉瘤患者易发生转移且临床预后较差，生存时间较短。骨肉瘤组织中mGluR4基因表现为高表达、低甲基化。mGluR4甲基化水平与肿瘤转移和复发以及生存时间密切相关。我们研究还发现：相对于人成骨细胞，mGluR4在人骨肉瘤细胞株中呈高表达。发现4条显著富集通路上的差异表达基因可能通过mGluR4参与骨肉瘤细胞的发生和进展过程。EGR1 和CTCF可能起肿瘤抑制因子的作用参与mGluR4基因的转录调控，并且mGluR4通过与下游的趋化因子和趋化因子受体的相互作用参与骨肉瘤的进展。. 本项目研究结果表明mGluR4基因的多态性对骨肉瘤遗传易感性和预后的评估具有一定参考价值，并且mGluR4的表达影响了骨肉瘤的增殖和预后。我们的研究成果为预测骨肉瘤的基因易感性和靶向治疗提供了证据。通过本项目的实施，培养了3名硕士研究生，为广西的骨肉瘤基础研究储备了人才。