冠心病新的长链非编码RNA loc338758生物学功能研究
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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RNA loc338758,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loc338758 mRNA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,loc338758 mRNA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RNA loc338758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 regulated gene transcriptional activity and could in cis inhibited adjacent ATP2 B1 gene expression. It was suggested that upregulation of lncRNA loc338758 might lead to endothelial cells dysfunction by negatively regulating the ATP2 B1 gene expression, thus participating in the pathogenesis of atherosclerosis or CAD.
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 regulated gene transcriptional activity and could in cis inhibited adjacent ATP2 B1 gene expression. It was suggested that upregulation of lncRNA loc338758 might lead to endothelial cells dysfunction by negatively regulating the ATP2 B1 gene expression, thus participating in the pathogenesis of atherosclerosis or CAD.
引文
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[15]Holdt LM, Hoffmann S, Sass K, et al. Alu elements in ANRIL non-coding RNA at chromosome 9p21 modulate atherogenic cell functions through trans-regulation of gene networks. PLoS Genet,2013,9:e1003588.
[16]Holdt LM, Beutner F, Scholz M, et al. ANRIL Expression Is Associated With Atherosclerosis Risk at Chromosome 9p21.Arterioscler Thromb Vasc Biol, 2010, 30(3):620-627.
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[18]Soltani B, Bodaghabadi N, Mahpour G, et al. Nanoformulation of curcumin protects HUVEC endothelial cells against ionizing radiation and suppresses their adhesion to monocytes:potential in prevention of radiation-induced atherosclerosis. Biotechnol Lett,2016,38:2081-2088.
[19]Jaipersad AS, Lip GY, Silverman S, et al. The role of monocytes in angiogenesis and atherosclerosis. J Am Coll Cardiol, 2014, 63:1-11.
[20]Pjanic M, Miller CL, Wirka R, et al. Genetics and Genomics of Coronary Artery Disease. Curr Cardiol Rep, 2016, 18:102.
[21]An D, Zhang J, Tang X, et al. Association of ATP2B1 common variants with asymptomatic intracranial and extracranial large artery stenosis in hypertension patients. Clin Exp Hypertens, 2018,14:1-7.
[22]Little R, Cartwright EJ, Neyses L, et al. Plasma membrane calcium ATPases(PMCAs)as potential targets for the treatment of essential hypertension. Pharmacol Ther, 2016, 159:23-34.
[23]Benetti F, Micetic I, Carsughi F, et al. Insights into the oligomerization process of the C-terminal domain of human plasma membrane Ca(2)+-ATPase. Arch Biochem Biophys, 2011,506:194-200.
[24]Xu J, Qian HX, Hu SP, et al. Gender-Specific Association of ATP2B1 Variants with Susceptibility to Essential Hypertension in the Han Chinese Population. Biomed Res Int, 2016, 2016:1910565.
[25]Little R, Zi M, Hammad SK, et al. Reduced expression of PMCA1is associated with increased blood pressure with age which is preceded by remodelling of resistance arteries. Aging Cell, 2017,16:1104-1113.
[26]Wang Y, Zhang Y, Li Y, et al. Common variants in the ATP2B1gene are associated with hypertension and arterial stiffness in Chinese population. Mol Biol Rep, 2013, 40:1867-1873.
[27]Xi B, Shen Y, Zhao X, et al. Association of common variants in/near six genes(ATP2B1, CSK, MTHFR, CYP17A1, STK39 and FGF5)with blood pressure/hypertension risk in Chinese children. J Hum Hypertens, 2014, 28:32-36.
[28]Ganesh SK,Tragante V.Guo W, et al. Loci influencing blood pressure identified using a cardiovascular gene-centric array. Hum Mol Genet, 2013, 22:1663-1678.
[29]Takeuchi F, Isono M, Yamamoto K, et al. Heterogeneous effects of association between blood pressure loci and coronary artery disease in east Asian individuals. Circ J, 2015, 79:830-838.
[30]Fontana V, McDonough CW, Gong Y, et al. Large-scale gene-centric analysis identifies polymorphisms for resistant hypertension. J Am Heart Assoc, 2014, 3:e001398.
[31]Weng L, Taylor KD, Chen YD, et al. Genetic loci associated with nonobstructive coronary artery disease in Caucasian women.Physiol Genomics, 2016,48:12-20.
[32]Tolle M, Klockl L, Wiedon A, et al. Regulation of endothelial nitric oxide synthase activation in endothelial cells by SI P1 and S1P3.Biochem Biophys Res Commun, 2016, 476:627-634.
[2]Hanifehpour R, Motevalli M, Ghanaati H, et al. Diagnostic Accuracy of Coronary Calcium Score Less than 100 in Excluding Coronary Artery Disease. Iran J Radiol, 2016, 13:e16705.
[3]马丽媛,吴亚哲,王文,等.中国心血管病报告2017.中国心血管杂志,2018, 1:3-6.
[4]Wang XB, Han YD, Cui NH, et al. Associations of lipid levels susceptibility loci with coronary artery disease in Chinese population. Lipids Health Dis, 2015, 14:80.
[5]Khera AV and Kathiresan S. Genetics of coronary artery disease:discovery, biology and clinical translation. Nat Rev Genet, 2017,18:331-344.
[6]Pathak LA, Shirodkar S, Ruparelia R, et al. Coronary artery disease in women. Indian Heart J, 2017, 69:532-538.
[7]Matsuzawa Y, Lerman A. Endothelial dysfunction and coronary artery disease:assessment, prognosis, and treatment. Coron Artery Dis, 2014, 25:713-724.
[8]Lu X, Wang L, Chen S, et al. Genome-wide association study in Han Chinese identifies four new susceptibility loci for coronary artery disease. Nat Genet, 2012, 44:890-894.
[9]Zhu J, Fu H, Wu Y, et al. Function of IncRNAs and approaches to IncRNA-protein interactions. Sci China Life Sci, 2013, 56:876-885.
[10]Chen LL. Linking Long Noncoding RNA Localization and Function. Trends Biochem Sci, 2016, 41:761-772.
[11]Tarighi S, Alipoor B, Zare A, et al. Association of the rs1870634Variant in Long Intergenic Non-protein Coding RNA 841 with Coronary Artery Disease:A GWAS-Replication Study in an Iranian Population. Biochem Genet, 2018, 56:522-532.
[12]Samani NJ, Erdmann J, Hall AS, et al. Genomewide association analysis of coronary artery disease. N Engl J Med, 2007, 357:443-453.
[13]Helgadottir A, Thorleifsson G, Manolescu A, et al. A common variant on chromosome 9p21 affects the risk of myocardial infarction. Science, 2007, 316(5830):1491-1493.
[14]McPherson R, Pertsemlidis A, Kavaslar N, et al. A common allele on chromosome 9 associated with coronary heart disease. Science,2007.316(5830):1488-1491.
[15]Holdt LM, Hoffmann S, Sass K, et al. Alu elements in ANRIL non-coding RNA at chromosome 9p21 modulate atherogenic cell functions through trans-regulation of gene networks. PLoS Genet,2013,9:e1003588.
[16]Holdt LM, Beutner F, Scholz M, et al. ANRIL Expression Is Associated With Atherosclerosis Risk at Chromosome 9p21.Arterioscler Thromb Vasc Biol, 2010, 30(3):620-627.
[17]Fujiwara A, Hirawa N, Fujita M, et al. Impaired nitric oxide production and increased blood pressure in systemic heterozygous ATP2B1 null mice. J Hypertens, 2014, 32:1415-1423; discussion1423.
[18]Soltani B, Bodaghabadi N, Mahpour G, et al. Nanoformulation of curcumin protects HUVEC endothelial cells against ionizing radiation and suppresses their adhesion to monocytes:potential in prevention of radiation-induced atherosclerosis. Biotechnol Lett,2016,38:2081-2088.
[19]Jaipersad AS, Lip GY, Silverman S, et al. The role of monocytes in angiogenesis and atherosclerosis. J Am Coll Cardiol, 2014, 63:1-11.
[20]Pjanic M, Miller CL, Wirka R, et al. Genetics and Genomics of Coronary Artery Disease. Curr Cardiol Rep, 2016, 18:102.
[21]An D, Zhang J, Tang X, et al. Association of ATP2B1 common variants with asymptomatic intracranial and extracranial large artery stenosis in hypertension patients. Clin Exp Hypertens, 2018,14:1-7.
[22]Little R, Cartwright EJ, Neyses L, et al. Plasma membrane calcium ATPases(PMCAs)as potential targets for the treatment of essential hypertension. Pharmacol Ther, 2016, 159:23-34.
[23]Benetti F, Micetic I, Carsughi F, et al. Insights into the oligomerization process of the C-terminal domain of human plasma membrane Ca(2)+-ATPase. Arch Biochem Biophys, 2011,506:194-200.
[24]Xu J, Qian HX, Hu SP, et al. Gender-Specific Association of ATP2B1 Variants with Susceptibility to Essential Hypertension in the Han Chinese Population. Biomed Res Int, 2016, 2016:1910565.
[25]Little R, Zi M, Hammad SK, et al. Reduced expression of PMCA1is associated with increased blood pressure with age which is preceded by remodelling of resistance arteries. Aging Cell, 2017,16:1104-1113.
[26]Wang Y, Zhang Y, Li Y, et al. Common variants in the ATP2B1gene are associated with hypertension and arterial stiffness in Chinese population. Mol Biol Rep, 2013, 40:1867-1873.
[27]Xi B, Shen Y, Zhao X, et al. Association of common variants in/near six genes(ATP2B1, CSK, MTHFR, CYP17A1, STK39 and FGF5)with blood pressure/hypertension risk in Chinese children. J Hum Hypertens, 2014, 28:32-36.
[28]Ganesh SK,Tragante V.Guo W, et al. Loci influencing blood pressure identified using a cardiovascular gene-centric array. Hum Mol Genet, 2013, 22:1663-1678.
[29]Takeuchi F, Isono M, Yamamoto K, et al. Heterogeneous effects of association between blood pressure loci and coronary artery disease in east Asian individuals. Circ J, 2015, 79:830-838.
[30]Fontana V, McDonough CW, Gong Y, et al. Large-scale gene-centric analysis identifies polymorphisms for resistant hypertension. J Am Heart Assoc, 2014, 3:e001398.
[31]Weng L, Taylor KD, Chen YD, et al. Genetic loci associated with nonobstructive coronary artery disease in Caucasian women.Physiol Genomics, 2016,48:12-20.
[32]Tolle M, Klockl L, Wiedon A, et al. Regulation of endothelial nitric oxide synthase activation in endothelial cells by SI P1 and S1P3.Biochem Biophys Res Commun, 2016, 476:627-634.