冠心病EAT中與脂聯(lián)素基因相關(guān)差異性microRNAs的篩選及生物信息學(xué)分析
發(fā)布時間：2018-08-21 10:41
【摘要】：目的篩選在冠狀動脈粥樣硬化性心臟病(coronary atherosclerotic heart disease,CAD)患者和非冠心病(non-CAD)患者心外膜脂肪組織(epicardial adipose tissue,EAT)中差異性表達(dá)的microRNAs(differential expression of microRNAs,DE-miRNAs),并通過生物信息學(xué)分析脂聯(lián)素基因(adiponectin gene,ADIPOQ)相關(guān)的DE-miRNAs可能涉及的蛋白及信號通路。方法1.收集CAD(34例)和non-CAD(16例)患者EAT和血液標(biāo)本,采用miRNA芯片技術(shù)篩選出EAT中DE-miRNAs;2.找出與ADIPOQ 3’-非編碼區(qū)(3’-untranslated regions,UTR)有互補結(jié)合位點或密切相關(guān)的DE-miRNAs,采用實時熒光定量PCR(Real-time quantitative PCR,qRT-PCR)進(jìn)行驗證;3.選取兩個miRNAs(上調(diào)和下調(diào)各一)進(jìn)行生物信息學(xué)分析——首先預(yù)測差異miRNAs的靶基因,然后利用DAVID軟件對靶基因進(jìn)行GO功能富集分析和KEGG通路分析,最后導(dǎo)入STRING數(shù)據(jù)庫繪制蛋白互作網(wǎng)絡(luò)圖。結(jié)果CAD與non-CAD組相比,EAT標(biāo)本中共篩選出29個DE-miRNAs(P0.05;差異倍數(shù)2),其中下調(diào)10個,上調(diào)19個。miR-371b-5p和miR-155-5p是與ADIPOQ3’-UTR有互補結(jié)合位點或密切相關(guān)的2條DE-miRNAs。qRT-PCR驗證結(jié)果顯示,CAD與non-CAD組相比,miR-371b-5p在病人EAT組織和血漿中表達(dá)均上調(diào)(P0.05);而miR-155-5p表達(dá)則均下調(diào)(P0.05)。生物信息學(xué)分析顯示,miR-371b-5p和miR-155-5p預(yù)測靶基因的GO主要富集在轉(zhuǎn)錄激活因子活性,RNA聚合酶II核心啟動子近端區(qū)域序列特異性DNA結(jié)合,染色質(zhì)結(jié)合,蛋白結(jié)合等分子功能以及RNA聚合酶II啟動子的轉(zhuǎn)錄調(diào)控,以DNA為模板的轉(zhuǎn)錄,蛋白磷酸化等生物過程上。KEGG通路富集分析結(jié)果顯示miR-371b-5p和miR-155-5p的共同相關(guān)的重要信號通路分別是脂肪細(xì)胞因子信號通路、雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)信號通路、缺氧誘導(dǎo)因子-1(Hypoxia inducible factor,HIF-1)信號通路以及TNF信號通路。STRING蛋白互作分析結(jié)果顯示:預(yù)測靶基因所編碼的蛋白質(zhì)中,ADIPOQ、LEP、AKT1、MAPK10、PPARGC1A、PRKAA1和SLC2A4這7種靶蛋白在維持網(wǎng)絡(luò)穩(wěn)定性與相互作用中起到關(guān)鍵性作用,尤其是ADIPOQ、LEP和AKT1處于網(wǎng)絡(luò)互作圖的核心區(qū)域。結(jié)論CAD與non-CAD患者比較,EAT和血漿中miRNAs表達(dá)存在差異性,其中miR-371b-5p表達(dá)均上調(diào),而miR-155-5p表達(dá)則均下調(diào)。預(yù)測的靶基因ADIPOQ,LEP和AKT1可能是miR-371b-5p和miR-155-5p所調(diào)控的下游靶基因,它們編碼的下游蛋白與其他相關(guān)蛋白可能是通過脂肪細(xì)胞因子信號通路,mTOR信號通路、TNF信號通路以及HIF-1信號通路等,在CAD形成、發(fā)展中起到調(diào)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.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R541.4
本文編號：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.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R541.4
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