【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)中差異性表達的microRNAs(differential expression of microRNAs,DE-miRNAs),并通過生物信息學分析脂聯素基因(adiponectin gene,ADIPOQ)相關的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和血液標本,采用miRNA芯片技術篩選出EAT中DE-miRNAs;2.找出與ADIPOQ 3’-非編碼區(3’-untranslated regions,UTR)有互補結合位點或密切相關的DE-miRNAs,采用實時熒光定量PCR(Real-time quantitative PCR,qRT-PCR)進行驗證;3.選取兩個miRNAs(上調和下調各一)進行生物信息學分析——首先預測差異miRNAs的靶基因,然后利用DAVID軟件對靶基因進行GO功能富集分析和KEGG通路分析,最后導入STRING數據庫繪制蛋白互作網絡圖。結果CAD與non-CAD組相比,EAT標本中共篩選出29個DE-miRNAs(P0.05;差異倍數2),其中下調10個,上調19個。miR-371b-5p和miR-155-5p是與ADIPOQ3’-UTR有互補結合位點或密切相關的2條DE-miRNAs。qRT-PCR驗證結果顯示,CAD與non-CAD組相比,miR-371b-5p在病人EAT組織和血漿中表達均上調(P0.05);而miR-155-5p表達則均下調(P0.05)。生物信息學分析顯示,miR-371b-5p和miR-155-5p預測靶基因的GO主要富集在轉錄激活因子活性,RNA聚合酶II核心啟動子近端區域序列特異性DNA結合,染色質結合,蛋白結合等分子功能以及RNA聚合酶II啟動子的轉錄調控,以DNA為模板的轉錄,蛋白磷酸化等生物過程上。KEGG通路富集分析結果顯示miR-371b-5p和miR-155-5p的共同相關的重要信號通路分別是脂肪細胞因子信號通路、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蛋白互作分析結果顯示:預測靶基因所編碼的蛋白質中,ADIPOQ、LEP、AKT1、MAPK10、PPARGC1A、PRKAA1和SLC2A4這7種靶蛋白在維持網絡穩定性與相互作用中起到關鍵性作用,尤其是ADIPOQ、LEP和AKT1處于網絡互作圖的核心區域。結論CAD與non-CAD患者比較,EAT和血漿中miRNAs表達存在差異性,其中miR-371b-5p表達均上調,而miR-155-5p表達則均下調。預測的靶基因ADIPOQ,LEP和AKT1可能是miR-371b-5p和miR-155-5p所調控的下游靶基因,它們編碼的下游蛋白與其他相關蛋白可能是通過脂肪細胞因子信號通路,mTOR信號通路、TNF信號通路以及HIF-1信號通路等,在CAD形成、發展中起到調控的作用。
[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.
【學位授予單位】：石河子大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R541.4

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