Elakkad Aliaa M, Abou-Aisha Khaled, Hassanein Sally I, Gad Mohamed Z
Faculty of Pharmacy and Biotechnology, The German University in Cairo.
Drug Discov Ther. 2017;11(4):193-197. doi: 10.5582/ddt.2016.01083.
This study investigated the role of the T-786C polymorphism (SNP) in the 5'-flanking sequence of the endothelial nitric oxide synthase gene (eNOS) on its expression level in vascular endothelium with the ultimate goal of shedding more light on the mechanisms by which genetic variations of eNOS might affect the vascular level of nitric oxide (NO). Sequences in the 5'-flanking region of eNOS gene were PCR-amplified using genomic DNA templates isolated from blood samples collected from cardiovascular disease (CVD) patients. Two sequence-versions carrying the same SNP site were used; a short (345 bp) and an extended one (1,594 bp), numbered relative to the translational start site. All sequences were cloned into a promoter-less vector (pGL3-basic), which carries the firefly luciferase gene as a reporter. Genotyping of the T-786C polymorphism was performed using Sanger sequencing of the insert region. Luminescence levels were then recorded 24-48 h after transfecting human endothelial cell line (EA.hy926). Three genotypes were identified in the subject samples; TT, TC, or CC. The highest expression levels associated with the TT genotype, followed by the TC genotype, then the CC genotype. The extended sequence version produced higher expression levels compared to the shorter version. Our results provide evidence that the T allele at the T-786C SNP site of the eNOS gene results in increased expression of the enzyme, and consequently might provide a protective mechanism from CVD. The extended promoter sequence of eNOS resulted in higher expression of the gene, suggesting the presence of some essential binding sites for transcription enhancing proteins.
本研究调查了内皮型一氧化氮合酶基因（eNOS）5'-侧翼序列中的T-786C多态性（单核苷酸多态性，SNP）对其在血管内皮中表达水平的作用，最终目的是更深入地了解eNOS基因变异可能影响一氧化氮（NO）血管水平的机制。使用从心血管疾病（CVD）患者采集的血样中分离的基因组DNA模板，通过聚合酶链反应（PCR）扩增eNOS基因5'-侧翼区域的序列。使用了携带相同SNP位点的两个序列版本；一个短序列（345 bp）和一个长序列（1,594 bp），相对于翻译起始位点进行编号。所有序列均克隆到一个无启动子载体（pGL3-basic）中，该载体携带萤火虫荧光素酶基因作为报告基因。使用插入区域的桑格测序法对T-786C多态性进行基因分型。然后在转染人内皮细胞系（EA.hy926）后24 - 48小时记录发光水平。在受试者样本中鉴定出三种基因型；TT、TC或CC。与TT基因型相关的表达水平最高，其次是TC基因型，然后是CC基因型。与较短版本相比，较长序列版本产生更高的表达水平。我们的结果提供了证据，表明eNOS基因T-786C SNP位点的T等位基因导致该酶表达增加，因此可能提供一种预防CVD的保护机制。eNOS的延长启动子序列导致该基因更高的表达，表明存在一些转录增强蛋白的重要结合位点。
