【作者】 李萍；

【导师】 樊华；

【作者基本信息】 中国医科大学 ， 内科学（血液内科）（专业学位）， 2019， 硕士

【摘要】 目的:利用TCGA和GEO数据库分析miR-193b-3p和miR-196b-5p在急性髓系白血病(AML)非M3发病机制中的作用。方法1.首先利用在线数据库GEO下载GSE10358,GSE30285,GSE37642芯片数据,利用R包获得共同差异表达的基因(DEGs),并对DEGs进行GO功能富集分析,KEGG通路富集分析和PPI蛋白质互作分2.然后利用在线数据库TCGA获得169个AML非M3样本的miRNA表达矩阵。用R软件包DEseq2获得差异miRNA并通过在线预测网站预测差异miRNA的靶基因,并进行GO功能富集分析,KEGG通路富集分析和PPI蛋白质互作分析;3.最后利用R软件包Venny获取差异miRNA靶基因与三个GSE基因芯片共同差异基因的交集基因,并构建miRNA-gene调控网络,选择P值较小和以交集基因为靶基因的差异miRNA进行后续分析,研究差异miRNA在非M3型AML中的发病机制。结果:1.从GEO数据库中获得GSE10358,GSE30285,GSE37642的微阵列数据,使用p<0.05和logFC的绝对值>1作为截止标准,我们从中分别提取了340,503,324个差异表达基因,综合生物信息学分析,共鉴定出95个共同差异表达的基因,包括56个上调基因和39个下调基因。GEO差异基因本体分析(GO分析)在生物过程组中,上调基因主要富集于免疫应答,抗原处理、MHC II类呈递肽或多糖抗原过程,防御反应,损伤反应,下调基因主要富集在血液凝固,蛋白水解,免疫应答,细胞活动。在分子功能组中,上调基因主要富集在MHC蛋白结合。在细胞成分组中,上调基因主要富集于细胞质膜,细胞质膜外侧,下调基因在细胞成分组中无基因;KEGG通路富集分析上调基因主要富集于细胞粘附分子(CAMs),免疫信号系统,病毒性心肌炎,抗原呈递和处理,下调基因主要富集于止血,补体和凝血级联。2.从TCGA数据库中获得186个样本分成两组非M3和M3,样本个数分别为169和17个。使用R软件包DEseq2进行差异分析共得到前体miRNA:538个,按照P<0.05和两倍差异的miRNA共得到上调miRNA77个,下调miRNA61个。差异miRNA靶基因进行本体分析(GO分析)显示靶基因主要参与转录调节、转录因子活性、DNA连接、调节RNA代谢过程、转录调控DNA过程、氮化合物代谢过程、高分子生物合成过程、基因表达等多种与肿瘤相关的生物学过程和分子功能;在KEGG通路富集分析中,靶基因显著富集于癌症通路、WNT信号通路、整合信号通路、ErbB信号通路、Ras信号通路、NGF信号通路、MAPK信号通路、急性髓系白血病和慢性粒细胞白血病等信号通路。结论:(一)上调的miR-193b-3p通过靶向LACM1基因,在非M3中发挥抑癌因子作用。(二)(1)下调的miR-196b-5p通过靶向PTPRG基因和RAS通路可促进AML的发生;(2)通过靶向基因PBX3和激活MAPK信号途径诱导癌基因发挥作用;(3)通过靶向基因PBX和HOX的相互作用发挥致癌因子的作用。更多还原

【Abstract】 Objective: Analysis of the role of miR-193b-3p and miR-196b-5p in the pathogenesis of acute myeloid leukemia(AML)non-M3 using TCGA and GEO databases.Methods:1.Firstly,online database GEO was used to download the chip data of GSE10358,GSE30285 and GSE37642,and the common differentially expressed genes(DEGs)were obtained by using the R package.GO function enrichment analysis,KEGG pathway enrichment analysis and PPI protein interaction analysis were performed on DEGs.2.Secondly,miRNA expression matrix of 169 AML non-m3 samples was then obtained by using online database TCGA.Differential miRNA was obtained by R software package DEseq2 and target genes of differential miRNA were predicted through online prediction websites.GO function enrichment analysis,KEGG pathway enrichment analysis and PPI protein interaction analysis were conducted.3.Finally,the R software package Venny was used to obtain the intersection genes of the differentially expressed miRNA target genes and the common differentially expressed genes of the three GSE gene chips,and to construct a mirna-gene regulatory network.The differentially expressed miRNA with small P value and the intersection gene as the target genes were selected for subsequent analysis,so as to study the pathogenesis of differentially expressed miRNA in non-m3-type AML.Results: 1.Microarray data of GSE10358,GSE30285,and GSE37642 were obtained from GEO database.Using p<0.05 and absolute value >1 of logFC as cut-off criteria,340,503,324 differentially expressed genes were extracted from them respectively.In the biological process group,up-regulated genes were mainly concentrated in immune response,antigen processing,MHC II presenting peptide or polysaccharide antigen process,defense response,injury response,and down-regulated genes were mainly concentrated in blood coagulation,protein hydrolysis,immune response,and cell activity.In the molecular functional group,the up-regulated genes were mainly concentrated in MHC protein binding.In the cell grouping,the up-regulated genes were mainly concentrated in the cytoplasmic membrane,while the down-regulated genes were not in the cytoplasmic membrane.KEGG pathway enrichment analysis up-regulated genes were mainly concentrated in cell adhesion molecules(CAMs),immune signaling system,viral myocarditis,antigen presentation and treatment,while down-regulated genes were mainly concentrated in hemostasis,complement and coagulation cascade.2.186 samples obtained from TCGA database were divided into two groups,non-m3 and M3,with 169 and 17 samples respectively.A total of 538 precursor mirnas were obtained by differential analysis using R software package DEseq2,and 77 mirnas were up-regulated and 61 mirnas were down-regulated according to P<0.05 and double differences.Differences of miRNA target gene ontology analysis(GO)analysis shows that main target genes involved in transcription regulation,activity of transcription factors,DNA connection,regulate RNA metabolism,nitrogen metabolism,transcriptional regulation,DNA process,polymer biosynthesis,gene expression,such as a variety of biological processes associated with cancer and molecular functions;In the KEGG pathway enrichment analysis,the target genes were significantly enriched in cancer pathway,WNT signaling pathway,integrated signaling pathway,ErbB signaling pathway,Ras signaling pathway,NGF signaling pathway,MAPK signaling pathway,acute myeloid leukemia and chronic myelogenous leukemia.Conclusion :(一)the up-regulated mir-1933-3p plays a role of tumor suppressor in non-m3 by targeting LACM1 gene.(二)(1)down-regulated mir-196b-5p can promote the occurrence of AML by targeting PTPRG gene and RAS pathway;(2)induce oncogenes to play a role by targeting PBX3 and activating MAPK signaling pathway;(3)play the role of carcinogenic factors by targeting the interaction of PBX and HOX genes.更多还原