CAJ | 학술논문

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.
목적 통과미진렬-비교기인조잡교(microarray-based comparative genomic hybridization,aCGH)방법검측인골육류기인조적고패수이상,탐토병험증골육류중요신호통로적유전학이상급기재골육류발생、발전중적작용.방법 응용aCGH검측10례신선골육류표본적기인고패수변화,연후이용기인화기인조경도백과전서통로분석방법(Kyoto Encyclopedia of Genes and Genomes,KEGG)학정골육류유관신호통로적기인개변,병채용형광원위잡교(fluorescence in situ hybridization,FISH)화면역조직화학염색(Immunohistochemistry,IHC)등방법진행험증.결과 대10례골육류표본적aCGH수거진행KEGG통로분석발현33조신호통로적분자유명현적기인고패수개변,20조신호통로적분자존재명현적기인확증,여혈관내피생장인자(VEGF)화mTOR통로;13조신호통로적분자존재명현적기인결실,여Wnt화Hedgehog통로.령외,환발현여세포-세포-기질상호작용상관적신호통로야존재명현적유전학개변,여CAMs화긴밀련접신호통로존재기인확증、점착련접통로존재기인결실.통과형광원위잡교적방법증실VEGF신호통로중적VEGFA기인명현확증,통과면역조직화학염색방법증실골육류중VEGFA단백고표체,병차여미혈관밀도긴밀상관.결론 골육류신호통로적유전학개변섭급VEGF、mTOR、Wnt、Hedgehog、CAMs、긴밀련접、점착련접신호통로급기타26조신호통로,저사신호통로적유전학개변가능여골육류적발생、발전유관,위골육류침대특정신호통로적파향치료제공료유력적분자유전학의거.
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.