老年性痴呆相关基因nicastrin启动子区单核苷酸多态性分析

Single Nucleotide Polymorphism in Promoter of Nicastrin Gene Related with Alzheimer's Disease

【目的】分析老年性痴呆(Alzheimer病)相关基因nicastrin启动子区单核苷酸多态性(SNP),探讨其基因表达可能的分子调控机制。【方法】扩增nicastrin基因ATG上游约1.0kp启动子区DNA片段,与萤火虫荧光素酶报告基因载体pGL3-Basic进行重组质粒的克隆与鉴定,确立启动子区候选功能SNP及其相应位点结合的转录调节因子。应用Lipofectamine 2000对包含功能SNP的重组质粒进行CRL-2137细胞瞬时转染,双荧光素酶相对活性分析及电泳迁移率变动分析。【结果】Nicastrin基因启动子区960 bp DNA片段内存在功能SNP_rs10752637-922 G→T,与其相应位点结合的转录因子为hepatic leukemia factor(HLF),SNP_rs10752637-922 T的序列结构对启动萤火虫荧光素酶报告基因的表达功能较SNP_rs10752637 -922 G的序列结构强(P<0.05,n=3)。【结论】Nicastrin基因核心启动子应该在960 bp DNA片段内,SNP_rs10752637-922 G→T可能是启动子区有功能位点突变,包含rs10752637-922 G/T的核苷酸序列结构可能通过与HLF在内的转录调节因子的作用来实现对nicastrin基因表达差异的调控。

[Objective] To analyze the single nucleotide polymorphism （SNP） in promoter of nicastrin gene related with Alzheimer＇s disease （AD） and investigate the potential molecule mechanism of genetic expression and regulation. [Methods] An about 1.0 kb fragment upstream to the start codon ATG of nicastrin gene was firstly isolated from human genomic DNA by PCR. The recombinant plasmid of the fragment and the promoterless pGL3-Basic firefly luciferase reporter vector were cloned and identified. Putative function SNP candidate and its corresponding transcription factor binding site （TFBS） in promoter region were definited. The recombinant plasmid containing the SNP was transfected transiently in CRL-2137 cell line used by Lipofectamine 2000. Relative luciferase activity （RLA） was measured and electrophoretic mobility shift assay （EMSA） was analyzed. [Results] There were function SNP_rs10752637-922 G→T in 960 bp fragment of the promoter region, and its corresponding TFBS was hepatic leukemia factor （HLF）. The expression level of the promoter containing SNP_rs10752637-922 T was higher than that of SNP_rs10752637-922 G （P 〈 0.05,n=3）. [Conclusion] It indicated that the core promoter of nicastrin gene should be within 960 bp fragment. The rs10752637-922 G to T might be the functional point mutation for nicastrin gene promoter, and the ribonucleotide sequence comprising rs10752637-922 G/T might carry out gene expression and regulation in promoter region by way of interacting with some transcription factor including HLF.