骨肉瘤重要信号通路的遗传学研究

 目的 通过微阵列-比较基因组杂交（microarray-based comparative genomic hybridization，aCGH）方法检测人骨肉瘤基因组的拷贝数异常，探讨并验证骨肉瘤重要信号通路的遗传学异常及其在骨肉瘤发生、发展中的作用。方法 应用aCGH检测10例新鲜骨肉瘤标本的基因拷贝数变化，然后利用基因和基因组京都百科全书通路分析方法（Kyoto Encyclopedia of Genes and Genomes，KEGG）确定骨肉瘤有关信号通路的基因改变，并采用荧光原位杂交（fluorescence in situ hybridization, FISH）和免疫组织化学染色（Immunohistochemistry, IHC）等方法进行验证。结果 对10例骨肉瘤标本的aCGH数据进行KEGG通路分析发现33条信号通路的分子有明显的基因拷贝数改变，20条信号通路的分子存在明显的基因扩增，如血管内皮生长因子（VEGF）和mTOR通路；13条信号通路的分子存在明显的基因缺失，如Wnt和Hedgehog通路。另外，还发现与细胞-细胞-基质相互作用相关的信号通路也存在明显的遗传学改变，如CAMs和紧密连接信号通路存在基因扩增、黏着连接通路存在基因缺失。通过荧光原位杂交的方法证实VEGF信号通路中的VEGFA基因明显扩增，通过免疫组织化学染色方法证实骨肉瘤中VEGFA蛋白高表达，并且与微血管密度紧密相关。结论 骨肉瘤信号通路的遗传学改变涉及VEGF、mTOR、Wnt、Hedgehog、CAMs、紧密连接、黏着连接信号通路及其他26条信号通路，这些信号通路的遗传学改变可能与骨肉瘤的发生、发展有关，为骨肉瘤针对特定信号通路的靶向治疗提供了有力的分子遗传学依据。

Genetic aberrations of key signaling pathways in human osteosarcoma

Objective To performed microarray-based comparative genomic hybridization (aCGH) detection and carried out pathway analysis to gain a systemic view on the pathway alterations of the genetically altered genes in human osteosarcoma. Methods aCGH experiments were carried on 10 fresh osteosarcoma samples to obtain recurrent copy number change pattern, then the samples were further subjected to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis to identify the altered pathways in the osteosarcoma. To validate the aberrations of these key pathways, the alterations of VEGF pathway were selected to confirm by the methods of fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) in formalin-fixed and paraffin-embedded (FFPE) osteosarcoma archival tissues. Results The KEEG analysis of aCGH data identified 33 genetically altered pathways in osteosarcomas. Among them 20 pathways were identified genetic amplifications, such as VEGF and mTOR signaling pathways. Thirteen pathways were genetic deletions, such as Wnt and Hedgehog signaling pathways. The genetic aberrations of cell-cell-matrix pathway such as CAMs, Adherens junction and Tight junction pathways implied the genetically alterations of these pathways which are associated with the tumor invasion and metastasis. Validation the aberrations of VEGF pathway showed that VEGFA gene was significantly amplified. The positive protein expression of VEGFA had a significant association with microvessel density (MVD). Conclusion There are genetic aberrations which involved the component genes of VEGF, mTOR, CAMs, Adherens junction, Wnt, Hedgehog and other 26 signaling pathways. The alterations of these pathways which are significantly associated with tumor invasion, metastasis and progression suggest that the genetic aberrations of these key pathways might contribute to the tumorigenesis and progression in human osteosarcoma, and provide molecular genetic evidence for targeted therapy.