高通量测序结合生物信息学分析鉴定microRNA在颅内动脉瘤中的潜在作用

Identification of the Potential Role of microRNA in Intracranial Aneurysms Based on High-throughput Sequencing Combined with Bioinformatics Analysis

目的通过二代测序技术以及生物信息学方法筛选在颅内动脉瘤(IA)中失调的miRNA,并探索其可能参与的生物学功能。方法在GEO数据库编号为GSE66239的公共数据集中筛选IA和颞浅动脉组织间差异表达的miRNA,另外收集2021-2022年牡丹江医学院附属红旗医院收治的因颅内动脉瘤破裂出血行开颅动脉瘤夹闭术患者的IA组织和颞浅动脉组织,进行miRNA测序分析,验证公共数据集的结果。通过miRDB数据库和miRWalk数据库对miRNA的靶基因进行预测,使用R软件包clusterProfiler进行miRNA靶基因的功能富集分析,使用STRING数据库进行蛋白-蛋白互作网络(PPI)分析,并通过Cytescape软件进行可视化,提取PPI网络中的核心基因。基于GSE122897数据集中的转录组数据,使用单样本基因集富集分析(ssGSEA)评估IA和对照组织免疫微环境中16种免疫细胞的浸润分数和12种免疫状态评分。结果在GSE66239数据集中,相比于正常颞浅动脉组织,IA组织中有54个miRNA上调,1010个miRNA下调;临床标本中,相比于正常颞浅动脉组织,IA组织中有23个miRNA上调,29个miRNA下调。hsa-miR-3176、hsa-miR-1246和hsa-miR-539-3P在GSE66239数据集及临床标本中均呈现出IA组织的低表达。在miRDB数据库和miRWalk数据库中分别预测到738个和116个hsa-miR-539-3P靶向的mRNA,交集后得到9个靶向mRNA,407个和711个hsa-miR-1246靶向的mRNA,交集后得到34个mRNA,482个和3486个hsa-miR-3176靶向的mRNA,交集后得到187个mRNA。功能富集分析显示hsa-miR-3176、hsa-miR-1246和hsa-miR-539-3P的靶基因主要与MAPK信号通路、FGF通路、CD8+T细胞受体下游通路、S1P通路、G蛋白信号通路、ERBB信号通路、白介素信号、神经递质释放等通路有关。由hsa-miR-3176,hsa-miR-1246和hsa-miR-539-3P靶基因所构成的PPI网络当中包含121个节点,117条互作关系;其中ERBB4、FGF1、CBL、GNAO1、GNAZ、PTPRT、KCNB1、KCNJ2、POLR2J2和CD247为PPI网络当中的Top10核心基因,这些核心基因均由hsa-miR-3176调控。功能富集分析结果显示,这些核心基因与ERBB信号通路、Rap1信号通路、MAPK信号通路、PI3K-AKT信号通路、CXCR4信号通路、CXCR3信号通路、CD8+T细胞受体信号通路等生物学过程相关。与颞浅动脉组织相比,IA当中CD8+T细胞、巨噬细胞、肥大细胞、中性粒细胞、辅助性T细胞、Th1细胞、TIL细胞以及调节性T细胞的浸润量增加,APC共刺激评分、APC共抑制评分、CCR评分、免疫检查点评分、HLA评分、促炎评分、副炎症评分、T细胞共刺激评分、T细胞共抑制评分、2型免疫反应评分均升高。相关性分析显示,核心基因与免疫细胞浸润以及免疫状态评分具有相关性。结论hsa-miR-3176,hsa-miR-1246和hsa-miR-539-3P在IA组织中呈低表达,且其低表达会导致颅内动脉瘤的发生。

Objective To screen the dysregulated miRNAs in intracranial aneurysms(IA)by next-generation sequencing and bioinformatics methods,and to explore their possible biological functions.Methods The differentially expressed miRNAs between IA and superficial temporal artery tissues were screened in the public data set numbered GSE66239 in the GEO database.In addition,IA tissues and superficial temporal artery tissues of patients with rupture and hemorrhage of intracranial aneurysm who underwent craniotomy aneurysm clipping in Hongqi Hospital Affiliated to Mudanjiang Medical University from 2021 to 2022 were collected for miRNA sequencing analysis to verify the results of the public data set.The target genes of miRNAs were predicted by miRDB database and miRWalk database.The functional enrichment analysis of miRNA target genes was performed using the R software package clusterProfiler.The protein-protein interaction network(PPI)analysis was performed using the STRING database,and visualized by Cytescape software to extract the core genes in the PPI network.Based on the transcriptome data in the GSE122897 dataset,single-sample gene set enrichment analysis(ssGSEA)was used to evaluate the infiltration scores of 16 immune cells and 12 immune status scores in the immune.Results In the GSE66239 dataset,54 miRNAs were up-regulated and 1010 miRNAs were down-regulated in IA tissues compared with normal superficial temporal artery tissues.In clinical specimens,compared with normal superficial temporal artery tissue,23 miRNAs were up-regulated and 29 miRNAs were down-regulated in IA tissue.hsa-miR-3176,hsa-miR-1246 and hsa-miR-539-3P showed low expression in IA tissues in GSE66239 dataset and clinical specimens.And 738 and 116 hsa-miR-539-3P-targeted mRNAs were predicted in the miRDB database and the miRWalk database,respectively,and 9 targeted mRNAs were obtained after intersection.Correspondingly,hsa-miR-1246 obtained 407 and 711 targeted mRNAs in the miRDB database and the miRWalk database,respectively,and 34 mRNAs were obtained after intersection.hsa-miR-3176 obtained 482 and 3486 targeted mRNAs in the miRDB database and the miRWalk database,respectively,and 187 mRNAs were obtained after intersection.Functional enrichment analysis showed that the target genes of the above three miRNA were mainly related to the MAPK signaling pathway,FGF pathway,CD8+T cell receptor downstream pathway,S1P pathway,G protein signaling pathway,ERBB signaling pathway,interleukin signaling,neurotransmitter release and other pathways.The PPI network composed of hsa-miR-3176,hsa-miR-1246 and hsa-miR-539-3P target genes contained 121 nodes and 117 interactions.Among them,ERBB4,FGF1,CBL,GNAO1,GNAZ,PTPRT,KCNB1,KCNJ2,POLR2J2 and CD247 are the top 10 core genes in the PPI network,and these core genes were regulated by hsa-miR-3176.Functional enrichment analysis showed that these core genes were related to ERBB signaling pathway,Rap1 signaling pathway,MAPK signaling pathway,PI3K-AKT signaling pathway,CXCR4 signaling pathway,CXCR3 signaling pathway,CD8+T cell receptor signaling pathway and other biological processes.Compared with superficial temporal artery tissue,the infiltration of CD8+T cells,macrophages,mast cells,neutrophils,helper T cells,Th1 cells,TIL cells and regulatory T cells in IA increased,APC co-stimulation score,APC co-inhibition score,CCR score,immune examination point score,HLA score,pro-inflammatory score,para-inflammatory score,T cell co-stimulation score,T cell co-inhibition score and type 2 immune response score increased.Correlation analysis showed that core genes were correlated with immune cell infiltration and immune status scores.Conclusion hsa-miR-3176,hsa-miR-1246 and hsa-miR-539-3P are lowly expressed in IA tissues,and their low expression will lead to the occurrence of intracranial aneurysms.