冠心病EAT中与脂联素基因相关差异性microRNAs的筛选及生物信息学分析
发布时间：2018-08-21 10:41
【摘要】：目的筛选在冠状动脉粥样硬化性心脏病(coronary atherosclerotic heart disease,CAD)患者和非冠心病(non-CAD)患者心外膜脂肪组织(epicardial adipose tissue,EAT)中差异性表达的microRNAs(differential expression of microRNAs,DE-miRNAs),并通过生物信息学分析脂联素基因(adiponectin gene,ADIPOQ)相关的DE-miRNAs可能涉及的蛋白及信号通路。方法1.收集CAD(34例)和non-CAD(16例)患者EAT和血液标本,采用miRNA芯片技术筛选出EAT中DE-miRNAs;2.找出与ADIPOQ 3’-非编码区(3’-untranslated regions,UTR)有互补结合位点或密切相关的DE-miRNAs,采用实时荧光定量PCR(Real-time quantitative PCR,qRT-PCR)进行验证;3.选取两个miRNAs(上调和下调各一)进行生物信息学分析——首先预测差异miRNAs的靶基因,然后利用DAVID软件对靶基因进行GO功能富集分析和KEGG通路分析,最后导入STRING数据库绘制蛋白互作网络图。结果CAD与non-CAD组相比,EAT标本中共筛选出29个DE-miRNAs(P0.05;差异倍数2),其中下调10个,上调19个。miR-371b-5p和miR-155-5p是与ADIPOQ3’-UTR有互补结合位点或密切相关的2条DE-miRNAs。qRT-PCR验证结果显示,CAD与non-CAD组相比,miR-371b-5p在病人EAT组织和血浆中表达均上调(P0.05);而miR-155-5p表达则均下调(P0.05)。生物信息学分析显示,miR-371b-5p和miR-155-5p预测靶基因的GO主要富集在转录激活因子活性,RNA聚合酶II核心启动子近端区域序列特异性DNA结合,染色质结合,蛋白结合等分子功能以及RNA聚合酶II启动子的转录调控,以DNA为模板的转录,蛋白磷酸化等生物过程上。KEGG通路富集分析结果显示miR-371b-5p和miR-155-5p的共同相关的重要信号通路分别是脂肪细胞因子信号通路、雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)信号通路、缺氧诱导因子-1(Hypoxia inducible factor,HIF-1)信号通路以及TNF信号通路。STRING蛋白互作分析结果显示:预测靶基因所编码的蛋白质中,ADIPOQ、LEP、AKT1、MAPK10、PPARGC1A、PRKAA1和SLC2A4这7种靶蛋白在维持网络稳定性与相互作用中起到关键性作用,尤其是ADIPOQ、LEP和AKT1处于网络互作图的核心区域。结论CAD与non-CAD患者比较,EAT和血浆中miRNAs表达存在差异性,其中miR-371b-5p表达均上调,而miR-155-5p表达则均下调。预测的靶基因ADIPOQ,LEP和AKT1可能是miR-371b-5p和miR-155-5p所调控的下游靶基因,它们编码的下游蛋白与其他相关蛋白可能是通过脂肪细胞因子信号通路,mTOR信号通路、TNF信号通路以及HIF-1信号通路等,在CAD形成、发展中起到调控的作用。
[Abstract]:Objective to screen the differentially expressed microRNAs (differential expression of microRNAs from epicardial adipose tissue (epicardial adipose tissue in patients with coronary atherosclerotic heart disease (coronary atherosclerotic heart disease) and non-coronary heart disease (non-CAD), and to analyze the adiponectin gene ADIPOQ phase by bioinformatics. Turning off DE-miRNAs may involve protein and signaling pathways. Method 1. EAT and blood samples were collected from 34 patients with CAD and 16 patients with non-CAD. DE-miRNAs2 in EAT was screened by miRNA microarray technique. The DE-miRNAss with complementary binding sites or closely related to 3'-untranslated regions were identified and verified by real-time fluorescence quantitative PCR (Real-time quantitative PCR qRT-PCR). Two miRNAs (one up-regulation and one down-regulation) were selected for bioinformatics analysis. The target genes of differential miRNAs were first predicted, then the target genes were analyzed by go function enrichment analysis and KEGG pathway analysis by DAVID software. Finally, the protein interaction network was drawn by importing STRING database. Results compared with non-CAD group, 29 DE-miRNAs (P0.05; difference multiple 2) were screened out in CAD, 10 of which were down-regulated. Upregulation of 19 miR-371b-5p and miR-155-5p were two complementary binding sites or closely related to ADIPOQ3'-UTR. The results showed that the expression of miR-371b-5p was up-regulated in EAT tissues and plasma compared with non-CAD group (P0.05), while miR-155-5p expression was down-regulated (P0.05). Bioinformatics analysis showed that the target genes of miR-371b-5p and miR-155-5p were mainly enriched in the transcriptional activator activity miR-155-5p polymerase II core promoter sequence specific DNA binding and chromatin binding. Protein binding and other molecular functions as well as the transcriptional regulation of RNA polymerase II promoter, and transcription using DNA as template. The enrichment analysis of .KEGG pathway in protein phosphorylation and other biological processes showed that the important signal pathways related to miR-371b-5p and miR-155-5p were adipocyte factor signaling pathway, rapamycin target protein (mammalian target of rapamycin mTOR signaling pathway, respectively. The results of HIF-1 signal pathway and TNF signaling pathway. STRING protein interaction analysis showed that the seven target proteins, MAPK10, PPARGC1, PRKAA1 and SLC2A4, play a key role in maintaining network stability and interaction. In particular, ADIPOQPLEP and AKT1 are at the core of network mapping. Conclusion there is a difference in the expression of miRNAs between CAD and non-CAD patients. The expression of miR-371b-5p is up-regulated and the expression of miR-155-5p is down-regulated. The predicted target genes ADIPOQOQOLEP and AKT1 may be downstream target genes regulated by miR-371b-5p and miR-155-5p. The downstream proteins and other related proteins encoded by them may be TNF- signaling pathway and HIF-1 signaling pathway through fatty cytokine signaling pathway. In the formation and development of CAD play a regulatory role.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R541.4
本文编号：2195449
[Abstract]:Objective to screen the differentially expressed microRNAs (differential expression of microRNAs from epicardial adipose tissue (epicardial adipose tissue in patients with coronary atherosclerotic heart disease (coronary atherosclerotic heart disease) and non-coronary heart disease (non-CAD), and to analyze the adiponectin gene ADIPOQ phase by bioinformatics. Turning off DE-miRNAs may involve protein and signaling pathways. Method 1. EAT and blood samples were collected from 34 patients with CAD and 16 patients with non-CAD. DE-miRNAs2 in EAT was screened by miRNA microarray technique. The DE-miRNAss with complementary binding sites or closely related to 3'-untranslated regions were identified and verified by real-time fluorescence quantitative PCR (Real-time quantitative PCR qRT-PCR). Two miRNAs (one up-regulation and one down-regulation) were selected for bioinformatics analysis. The target genes of differential miRNAs were first predicted, then the target genes were analyzed by go function enrichment analysis and KEGG pathway analysis by DAVID software. Finally, the protein interaction network was drawn by importing STRING database. Results compared with non-CAD group, 29 DE-miRNAs (P0.05; difference multiple 2) were screened out in CAD, 10 of which were down-regulated. Upregulation of 19 miR-371b-5p and miR-155-5p were two complementary binding sites or closely related to ADIPOQ3'-UTR. The results showed that the expression of miR-371b-5p was up-regulated in EAT tissues and plasma compared with non-CAD group (P0.05), while miR-155-5p expression was down-regulated (P0.05). Bioinformatics analysis showed that the target genes of miR-371b-5p and miR-155-5p were mainly enriched in the transcriptional activator activity miR-155-5p polymerase II core promoter sequence specific DNA binding and chromatin binding. Protein binding and other molecular functions as well as the transcriptional regulation of RNA polymerase II promoter, and transcription using DNA as template. The enrichment analysis of .KEGG pathway in protein phosphorylation and other biological processes showed that the important signal pathways related to miR-371b-5p and miR-155-5p were adipocyte factor signaling pathway, rapamycin target protein (mammalian target of rapamycin mTOR signaling pathway, respectively. The results of HIF-1 signal pathway and TNF signaling pathway. STRING protein interaction analysis showed that the seven target proteins, MAPK10, PPARGC1, PRKAA1 and SLC2A4, play a key role in maintaining network stability and interaction. In particular, ADIPOQPLEP and AKT1 are at the core of network mapping. Conclusion there is a difference in the expression of miRNAs between CAD and non-CAD patients. The expression of miR-371b-5p is up-regulated and the expression of miR-155-5p is down-regulated. The predicted target genes ADIPOQOQOLEP and AKT1 may be downstream target genes regulated by miR-371b-5p and miR-155-5p. The downstream proteins and other related proteins encoded by them may be TNF- signaling pathway and HIF-1 signaling pathway through fatty cytokine signaling pathway. In the formation and development of CAD play a regulatory role.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R541.4
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