SIRT2基因启动子在心肌梗死病人中遗传和功能变异研究

发布时间：2018-07-24 19:45

【摘要】：研究背景:急性心肌梗死(AMI)是冠状动脉性疾病(CAD)急性发作的一种表现形式,主要是由不稳定性动脉粥样硬化斑块破裂所致。目前已知关于动脉粥样硬化的病理机制主要涉及细胞炎症、氧化应激、血小板功能等多个代谢过程。尽管前期全基因组关联研究(GWAS)发现CAD的发生与遗传变异有关,但这些遗传变异仅能解释10%的CAD发生。目前认为低频率以及罕见的遗传变异可能与冠心病的发生有关。SIRT2属于sirtuin蛋白家族的一员,主要存在于哺乳动物体内,是一种高度保守依赖烟碱胺腺嘌呤二核苷酸(NAD+)的第Ⅲ类组蛋白去乙酰化酶。SIRT2在基因组稳定性、新陈代谢、炎症、氧化应激、自噬以及在调节血小板及血管内皮功能方面发挥着重要的作用。在基因表达过程中,启动子可调节基因表达水平变化,因此,本研究推测SIRT2基因的遗传变异在AMI的发病机制中可能存在重要的作用,力求通过研究SIRT2基因启动子区域的遗传变异所引起的基因表达水平的变化来探讨AMI的发生与SIRT2基因的关系。研究目的:首先在两组人群中通过测序探讨低频遗传变异以及单核苷酸多态性(SNPs)对AMI组和对照组人群发病的影响;其次通过构建携带SIRT2基因野生型及遗传变异位点启动子的pGL3-basic报告基因载体,检测遗传变异导致的基因表达水平的变化。通过对SIRT2基因启动子遗传及功能变异的研究,探讨SIRT2基因在AMI发病机制中的作用。研究方法:1、本研究最终纳入375例新发AMI病例和377例健康对照人群,分别收集两组人群临床基线资料并提取全基因组DNA。2、根据NCBI基因数据库提供的人SIRT2基因启动子序列设计引物,采用PCR方法扩增SIRT2基因启动子目的片段,直接测序后统计分析SIRT2的基因序列变异。3、将测序发现的携带SIRT2基因启动子序列变异位点及野生型目的基因片段构建到pGL3-basic报告基因载体,将构建好的pGL3-basic报告基因载体及内参质粒pRL-TK通过脂质体共转染HEK293及H9c2细胞,通过Promega双荧光报告基因分析仪分析SIRT2基因启动子的相对荧光素酶活性,检测基因遗传变异导致基因启动子转录活性的变化。研究结果:1、通过测序,共发现17个DNA序列变异(DSVs)位点,包括5个SNPs位点。其中发现3个新的杂合DSVs,分别为g.38900270AG、g.38899853CT和g.38900888_91delTAAA,仅存在于3个AMI病人中,在正常对照组中尚未发现。在对照组中发现5个新的DSVs,分别为g.38900562CT,g.38900413AC,g.38900030GA,g.38899925AC 和 g.38899852CT,而在 AMI 组中未发现上述DSVs。其余4个新的杂合突变位点和5个SNPs在对照组及AMI组中均有发现,但无统计学意义(P0.05)。2、成功构建pGL3-basic报告基因载体,分别为pGL3-WT(野生型SIRT2基因启动子)、pGL3-38900907G、pGL3-38900888_91del、pGL3-38900562T、pGL3-38900413C、pGL3-38900291G、pGL3-38900270G、pGL3-38900030A、pGL3-38899925C、pGL3-38899903C、pGL3-38899853T、pGL3-38899852T 和pGL3-38899781G。3、通过脂质体体外瞬时转染HEK293及H9c2细胞,检测携带不同DSVs的SIRT2基因启动子的相对荧光素酶表达活性。(1)在HEK293细胞中,仅在AMI组中发现DSVs可影响SIRT2基因启动子转录活性:g.38900888_91delTAAA(P0.05)和 g.38900270AG(P0.01)可显著降低SIRT2基因启动子的转录活性;g.38899853CT显著增加SIRT2基因启动子的转录活性(P0.01)。将仅在对照组中发现5个DSVs(g.38900562CT,g.38900413AC,g.38900030GA,g.38899925AC 和 g.38899852CT)转染至HEK293细胞后,尚未发现可显著改变SIRT2基因启动子的转录活性(P0.05)。同时 4 个 DSVs[g.38900907AG(rs4803006),g.38900291CG(rs2053071),g.38899903TC和g.38899781CG]均存在于对照组及AMI组中,将其相应的表达载体转染至HEK293细胞后,仍未发现可显著改变SIRT2基因启动子的转录活性(P0.05)。(2)将 AMI 组中发现的 g.38900888_91delTAAA 和 g.38900270AG 转染至H9c2细胞中,发现可显著降低SIRT2基因启动子的转录活性(P0.01);g.38899853CT转染后可显著增加SIRT2基因启动子的转录活性(P0.01)。在AMI组及对照组出现相似频率的位点g.38900907AG(rs4803006)和g.38900291CG(rs2053071)转染至H9c2细胞后,未发现可显著改变SIRT2基因启动子的转录活性(P0.05)。研究结论:通过对SIRT2基因启动子遗传及功能学变异的研究发现,在AMI病例中发现的低频序列变异位点可能会影响SIRT2基因的转录活性,导致其表达水平的变化,进而在心肌梗死的发生发展中起着重要的作用,为心肌梗死的预防和治疗提供了潜在的靶点。
[Abstract]:Background: acute myocardial infarction (AMI) is a manifestation of acute attack of coronary artery disease (CAD). It is mainly caused by the rupture of unstable atherosclerotic plaque. The pathological mechanism of atherosclerosis is known to be mainly involved in many metabolic processes, such as cell inflammation, oxygenation stress, platelet function, and so on. The whole genome association study (GWAS) found that the occurrence of CAD is associated with genetic variation, but these genetic variations can only explain 10% of the occurrence of CAD. Low frequency and rare genetic variation may be associated with the occurrence of coronary heart disease that.SIRT2 belongs to a member of the sirtuin family, which is mainly in mammals, and is highly conservative. The third class histone deacetylase.SIRT2 of nicotinamide adenine adenine dinucleotide (NAD+) plays an important role in genomic stability, metabolism, inflammation, oxidative stress, autophagy, and the regulation of platelet and vascular endothelial function. In the process of gene expression, promoter can regulate the change of gene expression level. Therefore, this study It is presumed that the genetic variation of SIRT2 gene may have an important role in the pathogenesis of AMI. To explore the relationship between the occurrence of AMI and the SIRT2 gene by studying the changes in the gene expression level of the genetic variation in the promoter region of the SIRT2 gene, the purpose of this study is to explore the low frequency inheritance by sequencing in two groups of people. The effect of mutation and single nucleotide polymorphism (SNPs) on the incidence of AMI and control groups; secondly, by constructing a pGL3-basic reporter vector carrying the SIRT2 gene and the promoter of the genetic variation site, the change of gene expression level caused by genetic variation was detected. The study on the genetic and functional variation of the promoter of the SIRT2 gene was carried out. The role of SIRT2 gene in the pathogenesis of AMI was investigated. 1. The study was finally included in 375 new AMI cases and 377 healthy controls. The clinical baseline data of two groups of people were collected and the whole genome DNA.2 was extracted respectively. The primers were designed according to the SIRT2 gene promoter sequence provided by the NCBI gene database, and the PCR method was used. The target fragment of SIRT2 gene promoter was amplified and sequenced to analyze the gene sequence variation.3 of SIRT2, and the sequence of mutations of the promoter sequence and the wild type target gene fragment of the SIRT2 gene were constructed to the pGL3-basic reporter gene carrier, and the constructed pGL3-basic reporter gene vector and the internal reference plasmid pRL-TK were used to pass the lipid. The plastids were co transfected with HEK293 and H9c2 cells, and the relative luciferase activity of SIRT2 promoter was analyzed by Promega double fluorescent reporter gene analyzer, and the changes of gene promoter transcriptional activity were detected by genetic variation. The results were as follows: 1, 17 DNA sequences (DSVs), including 5 SNPs loci, were found by sequencing. 3 new heterozygous DSVs, g.38900270AG, g.38899853CT and g.38900888_91delTAAA, were found only in 3 AMI patients, which were not found in the normal control group. 5 new DSVs were found in the control group, g.38900562CT, g.38900413AC, g.38900030GA, g.38899925AC and g.38899852CT in the AMI group. The remaining 4 new heterozygous mutation sites and 5 SNPs were found in the control group and the AMI group, but there was no statistical significance (P0.05).2. The pGL3-basic reporter gene carrier was successfully constructed, pGL3-WT (wild type SIRT2 gene promoter), pGL3-38900907G, pGL3-38900888_91del, pGL3-38900562T, pGL3-38900413C, pGL3-38900291G, etc. 38900030A, pGL3-38899925C, pGL3-38899903C, pGL3-38899853T, pGL3-38899852T and pGL3-38899781G.3 were transiently transfected into HEK293 and H9c2 cells in vitro by liposomes to detect the relative luciferase expression activity of the SIRT2 gene promoter with different DSVs. (1) in HEK293 cells, only in the AMI group could affect the promoter of the gene promoter. Transcriptional activity: g.38900888_91delTAAA (P0.05) and g.38900270AG (P0.01) significantly reduced the transcriptional activity of the SIRT2 gene promoter, and g.38899853CT significantly increased the transcriptional activity of the SIRT2 gene promoter (P0.01). Only 5 DSVs (g.38900562CT, g.38900413AC, g.38900030GA, P0.01) could be found in the control group. After 93 cells, there was no significant change in the transcriptional activity (P0.05) of the SIRT2 gene promoter, and 4 DSVs[g.38900907AG (rs4803006), g.38900291CG (rs2053071), g.38899903TC and g.38899781CG] all existed in the control group and the AMI group. After transfection of its corresponding expression vector to HEK293 cells, no significant changes were found in SIRT2 basis. The transcriptional activity of promoter (P0.05). (2) transfection of g.38900888_91delTAAA and g.38900270AG found in group AMI into H9c2 cells could significantly reduce the transcriptional activity of SIRT2 gene promoter (P0.01). G.38899853CT transfection could significantly increase the transcriptional activity of the SIRT2 gene promoter (P0.01). Similar to the AMI group and the control group. After transfection of frequency sites g.38900907AG (rs4803006) and g.38900291CG (rs2053071) to H9c2 cells, there was no significant change in the transcriptional activity of the SIRT2 gene promoter (P0.05). Conclusion: the study of the genetic and functional variation of the promoter of SIRT2 gene found that the low frequency sequence found in the AMI case may affect the mutation site in the AMI case. The transcriptional activity of SIRT2 gene leads to changes in its expression level and plays an important role in the occurrence and development of myocardial infarction, which provides potential targets for the prevention and treatment of myocardial infarction.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R542.22

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