摘要
目的中国人冠心病全基因组关联研究鉴定了4个新的冠心病易感区域,其中12q21.33区域中rs7136259位点附近有一个功能未知的长链非编码RNAloc338758,本研究旨在探索loc338758在动脉粥样硬化及冠心病发生中的生物学功能。方法应用实时荧光定量聚合酶链反应检测一般人群和冠心病患者、对照人群外周血单个核细胞RNA中loc338758及其邻近基因ATP2B1mRNA的表达水平;应用ENCODE数据库分析loc338758所在基因组片段的功能调控元件;应用双荧光素酶报告基因系统检测包含loc338758的基因组片段的转录调控作用;构建、包装过表达loc338758的腺病毒,在人脐静脉内皮细胞中过表达loc338758,应用实时荧光定量聚合酶链反应检测loc338758临近基因ATP2B1的mRNA表达水平,应用蛋白质印迹技术检测ATP2B1蛋白表达水平。结果在一般人群样本中loc338758mRNA水平与冠心病易感位点rs7136259的疾病风险等位基因T的基因型分布显著相关。与对照组相比,loc338758mRNA水平在冠心病病例组中显著上升,而ATP2B1基因mRNA表达水平在冠心病病例组中显著下降。ENCODE数据库分析显示在血管内皮细胞中loc338758所在基因组片段具有丰富的转录调控元件。双荧光素酶报告基因结果表明包含loc338758的基因组片段显著抑制基因的转录活性。在人脐静脉内皮细胞中腺病毒介导的loc338758过表达显著抑制ATP2B1基因mRNA和蛋白表达水平。结论冠心病易感区域12q21.33中新的长链非编码RNAloc338758具有抑制基因转录活性的作用,能够抑制临近基因ATP2B1的表达。提示loc338758可能通过负调控ATP2B1基因的表达,从而影响内皮细胞功能紊乱,参与动脉粥样硬化及冠心病的发生和发展。
Objective Genome wide association study(GWAS) in Han Chinese identified four new susceptibility loci for coronary artery disease(CAD). Long non-coding RNA loc338758 is located in the 12q21.33 susceptibility loci, near to the lead single nucleotide polymorphism(SNP)rs7136259, but the role and underlying mechanism of loc338758 in the pathogenesis of atherosclerosis or CAD remains unclear. This study aims to explore the preliminary biological function of loc338758 in the pathogenesis of atherosclerosis or CAD. Methods Real-time quantitative polymerase chain reaction(qRT-PCR) was used to detect loc338758 and ATP2 B1 mRNA levels in peripheral blood mononuclear(PBMCs) in a cohort and CAD cases and controls;ENCODE database was used to analyze the potential regulatory elements of genomic DNA containing loc338758;Dual luciferase reporter gene system was used to determine the effect of genomic fragments containing loc338758 on gene transcriptional activity;Human umbilical vein endothelial cells(HUVECs) were infected with adenovirus vector expressing loc338758(Ad-loc338758) and adenovirus expressing GFP(Ad-GFP) as a negative control, and qRT-PCR and Western blotting were used to detect ATP2 B1 mRNA and protein expression,respectively. Results The mRNA level of loc338758 was significantly correlated with the genotype distribution of rs7136259 T allele in the cohort.Compared with controls. loc338758 mRNA level was significantly higher in CAD patients, while ATP2 B1 mRNA level was significantly lower in CAD patients. ENCODE database analysis showed that genomic fragments containing loc338758 had abundant transcriptional regulatory elements in vascular endothelial cells;Dual luciferase reporter gene results showed that genomic fragments containing loc338758 could significantly and drastically inhibit gene transcriptional activity. Adenovirus-medicated loc338758 overexpression in HUVECs significantly inhibited ATP2 B1 mRNA and protein expression. Conclusion LncRNA loc338758 in 12 q21.33 susceptibility loci of CAD negatively regu
作者
朱慧娟
杨彬
李宏帆
李琳
王来元
ZHU Hui-juan;YANG Bin;LI Hong-fan;LI Lin;WANG Lai-yuan(Department of Epidemiology,State Key Laboratory of Cardiovascular Disease,Fuwai Hospital,National Center for Cardiovascular Disease,Chinese Academy of Medical Science and Peking Union Medical College,Beijing 100037,China)
出处
《中国分子心脏病学杂志》
CAS
2019年第2期2839-2843,共5页
Molecular Cardiology of China
基金
中国医学科学院医学与健康科技创新工程(2016-I2M-1-009)
国家自然科学基金(91439202、81270334)
