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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 of novel glioma-associated LNX gene in different grades of astrocytomas and its molecular mechanisms involved in glioma signaling pathways. Methods The fetal brain cDNA library was constructed and sequenced to obtain the full-length LNX gene. The human multi-tissue library (MTC) was used as a template to study the normal human tissue distribution of LNX. 13,939 human gene expression profiling chips were used to detect LNX gene in brain star The expression of LNX gene in different grades of gliomas was confirmed by Northern blotting. The yeast two-hybrid system was used to study the proteins that interact with the new gene of LNX. The co-immunoprecipitation method was used to confirm the relationship between LNX, Numb protein and SK IP protein interaction. Results The cloned LNX gene in human fetal brain library was about 3.7 kb in length and contained 1 899 bp open reading frame encoding a 632 amino acid protein with a relative molecular mass of 70 000 with accession number AF237782. The new LNX gene was expressed in brain, kidney and pancreas The expression of LNX in glioma tissues was lower than that in the control group. The expression of LNX in glioma tissues was significantly correlated with tumorigenesis. The yeast two-hybrid screening of LNX gene and tumor Notch The interaction between Numb and SK IP proteins in the signaling pathway and co-immunoprecipitation revealed that the new LNX gene interacts with the SK IP protein and may affect Numb subcellular localization, thereby affecting the Notch signaling pathway Numb binding site. Conclusion The expression microarray can rapidly and efficiently lock down the genes closely related to glioma and find tumor markers. The new gene of LNX is closely related to glioma, which is involved in the development of glioma through the Notch signaling pathway and may become a glioma Potential therapeutic target.