人LNX新基因克隆及组织分布和在脑胶质瘤中的表达  

Molecular cloning,tissue distribution,and expression of a novel human gene,LNX gene,in gliomas

作　　者：陈菊祥[1] 卢亦成[1] 胡国汉[1] 孙克华[1] 骆纯[1] 赵炜[2] 吴海[2] 谢毅[2] 

机构地区：[1]第二军医大学附属长征医院神经外科,上海神经外科研究所,200003 [2]复旦大学生命科学院遗传工程国家重点实验室

出　　处：《中华医学杂志》2005年第38期2704-2710,共7页National Medical Journal of China

基　　金：国家自然科学基金资助项目(30471777)

摘　　要：目的探讨人脑胶质瘤相关LNX新基因克隆、组织分布和在不同级别星形细胞瘤中的表达及其参与胶质瘤信号途径的分子机制。方法构建胎脑cDNA文库并测序获得全长LNX新基因;人多组织文库(MTC)为模板研究LNX的正常人体组织分布;应用含13 939种人类基因表达谱芯片检测LNX新基因在脑星形细胞瘤中的表达;Northern杂交验证LNX新基因在不同级别胶质瘤中的表达;酵母双杂交研究与LNX新基因相互作用的蛋白,应用免疫共沉淀方法验证LNX与Numb蛋白和SK IP蛋白的相互作用。结果人胎脑文库中克隆的LNX新基因长约3.7 kb,含1 899 bp开放阅读框,编码632氨基酸蛋白,相对分子质量约70 000,登录号AF237782;LNX新基因在脑、肾和胰腺中表达较高,在心、胎盘、肺、肝、脾、胸腺和前列腺中也有表达;但LNX在胶质瘤组织中表达均降低,与肿瘤发生密切相关;酵母双杂交筛选到LNX新基因与肿瘤Notch信号途径中的Numb和SK IP蛋白有相互作用,免疫共沉淀发现LNX新基因与SK IP蛋白相互作用,可影响Numb的亚细胞定位,从而影响Notch信号途径Numb结合位点。结论表达谱芯片可快速高效锁定与胶质瘤密切相关的基因和寻找肿瘤标志物,LNX新基因与胶质瘤密切相关,通过Notch信号传导途径参与脑胶质瘤发生发展,可成为胶质瘤潜在的治疗靶标。Objective To investigate the cloning, tissue distribution, and expression pattern of the novel human gene, LNX gene, and characterize its molecular mechanism in all grades of human gliomas. Methods A novel human gene, LNX gene, was isolated from a fetal brain cDNA library. The expression pattern of LNX gene in 16 normal tissues was examined by MTC panel （Clontech）. Microarray were adopted and hybridized with LNX probes to examine the expression of the novel gene in 18 samples of glioma. Northern hybridization was used for verifying the expression of LNX gene in gliomas. Two-hybrid screen in yeast was used to identify human LNX interacting proteins. Pull-down assays and co-immunoprecipitation were transfected in HEK293 cells according to the lipofectAMINE protocol. Results A 3.7 kb eDNA containing an open reading frame （ORF） of 1 899 bp and a putative 632 amino acids protein was isolated, which was located on 4q12. cDNA microarray showed that LNX was down-regulated in all 18 glioma samples and it was testified by Northern blotting. The MTC panel showed a ubiquitous expression pattern which highly expressed in the brain, kidney, and pancreas, however, weakly expressed in the heart, lung, etc of the adult patients. The two - hybrid screen in yeast revealed that LNX interacted with Ski interacting protein （SKIP） via PDZ domains. Co-immunoprecipitation suggested that LNX interacted with SKIP in HEK293 cells and could affect the subcellular localization of Numb, which indicated that LNX might function as a molecular anchor localizing Numb to the subcellular site of its interaction with Notch. Conclusion eDNA microarray technology is a powerful technique in screening and locking differentially expressed genes in gliomas. LNX is closely related to human gliomas and plays an important role in gliomas by notch signal approach.

关 键 词：脑肿瘤 LNX基因 酵母双杂交 免疫沉淀 人脑胶质瘤 组织分布 基因克隆 NOTCH信号途径 NORTHERN杂交 蛋白相互作用 

分 类 号：R739.4[医药卫生—肿瘤]