小儿特发性扩张型心肌病核心基因鉴定和免疫浸润分析尤红俊1,赵倩倩2,苟棋玲1,董梦雅1摘要目的:利用生物信息学分析筛选小儿特发性扩张型心肌病(PIDC)的核心基因,并进行免疫浸润分析㊂方法:使用R语言对来自基因表达综合数据库(GEO)的PIDC转录谱进行差异分析㊂利用R语言对差异表达基因(DEGs)进行基因本体(GO)功能富集分析和京都基因与基因组百科全书(KEGG)通路富集分析㊂通过STRING数据库和CytoHubba构建蛋白-蛋白互作(PPI)网络及鉴定核心基因㊂利用比较毒理基因组学数据库(CTD)分析核心基因与心肌病㊁心力衰竭风险的关系㊂利用单样本基因集富集分析(ssGSEA)计算PIDC中28种免疫细胞在免疫浸润微环境中的比例㊂结果:PIDC左心室有137个DEGs,主要参与对外部刺激的正向调节㊁炎症反应的调节㊁中性粒细胞的激活和介导的免疫㊁细胞-基质黏附的正向调节㊁细胞外结构㊁含胶原的细胞外基质等㊂DEGs参与细胞凋亡㊁胰岛素抵抗㊁花生四烯酸代谢和缺氧诱导因子1(HIF-1)信号通路等㊂鉴定的核心基因为信号转导和转录激活因子3(STAT3)㊁CD68㊁高亲和力IgE受体γ亚基基因(FCER1G)㊁细胞周期素D1(CCND1)和CD163,其与心肌病㊁心力衰竭关联紧密㊂PIDC左心室组织中央记忆型CD8+T细胞的含量较高;活化的树突样细胞㊁骨髓来源的抑制性细胞㊁自然杀伤T细胞㊁浆细胞样树突状细胞㊁滤泡辅助性T细胞的含量较低㊂结论:炎症及免疫反应在PIDC的发病机制中发挥着重要作用,5个核心基因和6种免疫细胞与PIDC发生发展密切相关㊂关键词扩张型心肌病;差异表达基因;生物信息学;核心基因;免疫浸润d o i:10.12102/j.i s s n.1672-1349.2023.20.005Identification of Key Genes and Analysis of Immune Infiltration in Pediatric Idiopathic Dilated CardiomyopathyYOU Hongjun,ZHAO Qianqian,GOU Qiling,DONG MengyaShaanxi Provincial People's Hospital,Xi'an710068,Shaanxi,ChinaCorresponding Author DONG Mengya,E-mail:****************Abstract Objective:To screen key genes of pediatric idiopathic dilated cardiomyopathy(PIDC)using bioinformatics analysis and perform analysis of immune infiltration.Methods:Differential analysis of PIDC transcriptional profiles from Gene Expression Omnibus (GEO)was performed using the R language.Gene Ontology(GO)functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis of differentially expressed genes(DEGs)were conducted by R language.The STRING database and CytoHubba were used to construct the protein-protein interaction(PPI)network and identify the key genes.The association between key genes and risk of cardiomyopathy and heart failure was analyzed using the comparative toxicogenomics database(CTD).Single sample gene set enrichment analysis(ssGSEA)was used to calculate the proportion of28kinds of immune cells in immunoinfiltrated microenvironment with PIDC.Results:There were137DEGs in the left ventricular tissue of PIDC,which were mainly involved in the positive regulation of external stimuli,the regulation of inflammatory response,the activation and mediated immunity of neutrophils,the positive regulation of cell-matrix adhesion,the extracellular structure,and the extracellular matrix containing collagen.DEGs were involved in apoptosis,insulin resistance,arachidonic acid metabolism,and hypoxia-inducible factor-1(HIF-1) signaling pathway.The identified key genes were signal transducer and activitor of transcription3(STAT3),CD68,high-affinity IgE receptor gamma subunit gene(FCER1G),cyclinD1(CCND1),and CD163,which were closely associated with cardiomyopathy and heart failure.The content of central memory CD8+T cell was increased in the left ventricular tissue of PIDC,while the contents of activated dendritic cell,myeloid-derived suppressor cell,natural killer T cell,plasmacytoid dendritic cell,and follicular helper T cells were decreased.Conclusion:Inflammation and immune response played an important role in the pathogenesis of PIDC.Five key genes and six kinds of immune cells were closely related to the occurrence and development of PIDC.Keywords pediatric idiopathic dilated cardiomyopathy;differentially expressed genes;bioinformatics;key gene;immune infiltration作者单位 1.陕西省人民医院(西安710068);2.中国人民解放军空军军医大学第一附属医院通讯作者董梦雅,E-mail:****************引用信息尤红俊,赵倩倩,苟棋玲,等.小儿特发性扩张型心肌病核心基因鉴定和免疫浸润分析[J].中西医结合心脑血管病杂志,2023,21(20): 3703-3710.特发性扩张型心肌病(pediatric idiopathic dilatedcardiomyopathy,PIDC)是儿童常见的心肌病,是造成儿童心力衰竭的主要原因之一,同时导致恶性心律失常㊁血栓栓塞和猝死在内的一系列不良结局㊂目前小儿扩张型心肌病(dilated cardiomyopathy,DCM)的早期诊治水平不断提高,但病因不明㊁缺乏精准治疗,此类患儿预后不容乐观[1-2]㊂本研究基于基因表达综合数据库(gene expression omnibus,GEO)中PIDC的二代测序数据,寻找疾病的差异表达基因(differentially expressed genes,DEGs),通过基因本体(Gene Ontology,GO)功能富集分析和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路富集分析探讨发病机制,并构建蛋白-蛋白互作(protein-protein interaction,PPI)网络,筛选其中的核心基因,整合其与心肌病及心力衰竭风险的关系,辅以免疫细胞浸润分析,寻找疾病发生发展过程中的关键基因,为疾病的精准靶向治疗提供依据㊂1资料与方法1.1数据下载及预处理在GEO[3-4]中搜索 pediatric idiopathic dilated cardiomyopathy ,选取数据集GSE99321,其由美国科罗拉多大学药理学系Tatman P等科研人员免费提供,采用GPL16791Illumina HiSeq2500(Homo sapiens)为平台,收录14例小儿左心室组织二代测序数据,其中7例小儿PIDC作为试验组,另7例无心力衰竭小儿作为对照组㊂数据进行归一化预处理㊂1.2差异表达分析采用统计软件R语言(版本4.1.1) DESeq2 [5]包进行差异基因表达分析㊂以|log2(fold change)|ȡ1, P<0.05为DEGs的筛选条件㊂运用R语言ggplot2和pheatmap包绘制火山图和热图,进行DEGs可视化分析㊂1.3GO功能富集和KEGG通路富集分析使用R语言clusterProfiler和org.Hs.eg.db (Homo sapiens)包对DEGs进行GO功能富集分析和KEGG通路富集分析[6-7]㊂GO富集分析分别在生物过程(biological process,BP)㊁细胞组分(cellular component,CC)和分子功能(molecular function,MF)三方面对基因进行功能注释[8]㊂KEGG分析展示基因参与的信号通路㊂利用R语言ggplot2包将结果可视化㊂以P<0.05为差异有统计学意义㊂1.4PPI网络分析将DEGs导入STRING(Search Tool for the Retrieval of Interacting Genes,/cgi/ input.pl),进行PPI网络分析[9]㊂设定可信度confidence为0.4,输出TSV格式文件,用于Cytoscape分析[10]㊂1.5核心基因筛选使用Cytoscape3.7.3软件插件CytoHubba筛选出PPI网络中的核心基因㊂CytoHubba利用12种评分策略剖析PPI网络,包括最大相关准则(maximum correlation criterion,MCC)㊁度值(Degree值)等,该12种拓扑算法无优劣之分[11]㊂MCC代表基因/蛋白间最大相关性标准,以MCC值排序,居前5位的基因为PPI网络的核心基因㊂1.6利用CTD数据库评估核心基因与心肌病和心力衰竭的关联性利用比较毒理基因组学数据库(comparative toxicogenomics database,CTD)(/)分析核心基因与心肌病及心力衰竭风险的关系㊂CTD 整合了化合物-基因/蛋白质相互作用㊁化合物-疾病和基因-疾病关系的信息,探索与疾病机制相关的假设[12]㊂1.7免疫细胞浸润分析单样本基因集富集分析(single sample gene set enrichment analysis,ssGSEA)[13]根据表达谱计算每个基因的rank值,分析得到每个样本的免疫细胞的相对含量,有助于了解疾病状态下组织中所有免疫细胞的浸润情况,探讨其与疾病转归的关系㊂采用ssGSEA算法分析PIDC中28种免疫细胞在免疫浸润微环境中的比例㊂运用R语言pheatmap包和vioplot 包绘制热图和小提琴图进行可视化免疫细胞浸润分析㊂以P<0.05为差异有统计学意义㊂2结果2.1基因差异表达分析对原始矩阵数据进行归一化处理(见图1A㊁图1B)后,与对照组比较,PIDC左心室组织中有137个DEGs,其中70个表达上调㊁67个表达下调㊂对DEGs 绘制火山图进行可视化分析(见图1C)㊂取差异显著前100个基因绘制热图(见图1D)㊂图1PIDC基因差异表达分析(A为原始矩阵数据归一化处理前;B为原始矩阵数据归一化处理后;C为基因差异表达分析火山图;D为基因差异表达分析热图㊂其中红色表示上调;蓝色表示下调)2.2DEGs富集分析GO功能富集分析显示:DEGs主要参与对外部刺激的正向调节㊁炎症反应的调节㊁中性粒细胞的激活和介导的免疫㊁细胞-基质黏附的正向调节㊁细胞外结构㊁含胶原的细胞外基质等㊂详见图2A㊂KEGG通路富集分析显示:DEGs参与细胞凋亡㊁胰岛素抵抗㊁花生四烯酸代谢和缺氧诱导因子1(hypoxia-inducible factor1,HIF-1)信号通路等㊂详见图2B㊂图2GO功能富集和KEGG通路富集分析气泡图(A为GO富集分析;B为KEGG富集分析)2.3PPI网络分析及核心基因筛选对DEGs进行PPI网络分析,将离散节点隐去后,构建的初始PPI网络中节点(基因/蛋白)135个㊁边(蛋白互作关系)186条,平均节点度值为2.76,PPI富集P值<1.0e-16㊂输出TSV格式文件并导入Cytoscape软件,以CytoHubba插件进行拓扑计算,取MCC排名居前5位的基因作为核心基因,即信号转导与转录激活因子3(signal transducer and activitor of transcription 3,STAT3)㊁CD68㊁高亲和力IgE受体γ亚基基因(high-affinity IgE receptor gamma subunit gene,FCER1G)㊁细胞周期蛋白D1(cyclinD1,CCND1)和CD163㊂亮色标记的节点为核心基因,颜色深浅代表MCC值大小,共36个节点㊁100个边,详见图3A㊂以分组比较呈现两组5个核心基因的差异表达,详见图3B ㊂图3差异基因构成的PPI网络中鉴定核心基因及分组比较(A为鉴定核心基因;B为分组比较柱状图㊂*P<0.05,**P<0.01㊂色彩鲜明的节点为5个核心基因)2.4核心基因与心肌病㊁心力衰竭的关系核心基因与心肌病㊁心力衰竭的相互作用评分分析提示,5个核心基因STAT3㊁CD68㊁FCER1G㊁CCND1和CD163与心肌病㊁心力衰竭关联紧密㊂详见图4㊂可见核心基因与心肌病㊁心力衰竭关联性得分情况,得分越高提示关联性越大㊂图4核心基因与心肌病或心力衰竭关联性作用评分柱状图2.5免疫细胞浸润分析与对照组比较,试验组左心室组织中央记忆型CD8+T细胞含量较高,活化的树突样细胞㊁骨髓来源的抑制性细胞㊁自然杀伤T细胞㊁浆细胞样树突状细胞㊁滤泡辅助性T细胞含量较低,差异均有统计学意义(P<0.05)㊂详见图5㊁图6㊂图中红色表示上调,蓝色表示下调㊂图5PIDC免疫细胞浸润分析热图图6PIDC免疫细胞浸润分析小提琴图3讨论PIDC具有较高的发病率㊁死亡率,是小儿心脏移植的主要原因之一,严重威胁儿童身心健康㊂该病发病机制复杂,尚未明确,因而治疗难度较大㊂本研究基于生物信息学分析,探讨免疫相关的分子机制,并筛选出核心基因及浸润的免疫细胞,从而为探讨疾病机制㊁寻求治疗靶点提供理论依据㊂首先,本研究发现PIDC左心室组织中存在137个DEGs㊂其次,GO功能富集和KEGG通路富集分析发现DEGs的功能主要集中在对外部刺激的正向调节㊁炎症反应的调节㊁中性粒细胞的激活和介导的免疫㊁细胞-基质黏附的正向调节㊁细胞外结构㊁含胶原的细胞外基质等;这些差异基因主要富集在细胞凋亡㊁胰岛素抵抗㊁花生四烯酸代谢和HIF-1信号通路等㊂免疫介导的炎症损伤是DCM发生发展中的关键过程之一[14]㊂已有研究报道,炎症反应由凋亡分子Fas配体(Fas ligand,FasL)通过心肌细胞和成纤维细胞中的细胞外信号调节激酶1/2(extracellular-signal-regulated kinase1/2,ERK1/2)被激活,最终诱导DCM 和心力衰竭的发生㊂阻断ERK1/2通路㊁抑制炎症等可阻止疾病的进展[15]㊂有研究显示,DCM中炎症趋化因子水平显著升高[16]㊂中性粒细胞作为炎症反应的重要参与者,其水平的高低与DCM心力衰竭的严重程度相关[17]㊂心脏细胞外基质是一个动态分子网络,为心脏功能提供结构支撑㊂细胞-基质相互作用的改变㊁细胞外基质重塑被认为是心肌扩张过程中细胞重构的重要组成部分[18-19]㊂细胞外基质重塑通过影响细胞归巢㊁成纤维细胞激活㊁局部黏附蛋白表达参与DCM等,导致心力衰竭发生发展[20]㊂同时本研究结果显示,细胞凋亡是PIDC发生发展过程中的关键致病因素之一㊂转录因子p53是人体重要的管家基因,可维持正常的心脏结构和生理功能㊂已有研究报道,p53水平升高促进DCM中的心脏重构,这可能是通过诱导细胞凋亡㊁细胞周期停滞实现的[21]㊂有研究显示,基因PPP1R13L双等位变异与PIDC强烈相关,而PPP1R13L的功能是编码p53蛋白的促凋亡蛋白抑制剂[22]㊂阿霉素作为一种化疗药物,可提高肿瘤病人生存率,但其诱导DCM的作用也有报道,认为这种不良反应是阿霉素通过增加线粒体膜通透性,进而增加心肌细胞凋亡实现的[23]㊂本研究结果显示,胰岛素抵抗在PIDC中有致病作用㊂胰岛素抵抗通过增加游离脂肪酸氧化,造成心肌细胞能量代谢障碍,促进DCM的形成;反之,DCM通过促炎细胞因子释放㊁儿茶酚胺等激素释放,交感神经系统的激活,进一步加重胰岛素抵抗[24]㊂上述研究均提示,干预细胞凋亡和胰岛素抵抗可能成为PIDC的治疗方向㊂有研究显示,花生四烯酸CYP450酶代谢的产物可能在克山病和DCM的发病机制中发挥着关键作用,如CYP2C19基因上调和蛋白表达频繁可能是一种保护性补偿反应,而CYP1A1可能加重心肌损伤[25]㊂本研究富集分析结果显示,差异基因显著参与花生四烯酸代谢通路,提示该通路可能作为PIDC心力衰竭治疗的靶点[26]㊂HIF-1是缺氧/缺血血管反应的主要调节因子,驱动数百个基因的转录激活,涉及血管反应㊁血管生成及骨髓源性血管生成细胞的动员和归巢[27]㊂特发性扩张型心肌病表现为血管生成缺陷,且心外膜血管疏松与HIF-1增加相关[28]㊂本研究发现多个基因以下调方式参与HIF-1信号通路中,提示该通路可能被抑制㊂干预HIF-1信号通路,可影响心肌血管生成,可能是PIDC的治疗靶点之一㊂其次,本研究通过PPI网络分析筛选了5个与心肌病及心力衰竭关系密切的核心基因㊂STAT3是对心脏具有保护作用的转录因子,STAT3的激活通过增强心肌细胞的预适应和后适应㊁抗炎㊁诱导生存基因表达㊁维持线粒体功能等多种方式发挥心脏保护作用㊂既往研究显示,STAT3通路的下调可诱发DCM的心肌不良重构[29]㊂有研究表明,心肌细胞特异性STAT3通过维持肌萎缩蛋白和α-肌聚糖的表达水平,防止扩张表型的发生和发展[30]㊂本研究发现STAT3的水平在PIDC中是显著下调的㊂CD68是巨噬细胞的标志,目前关于其在心肌病和心力衰竭中作用的研究结果存在争议㊂有研究显示,巨噬细胞作为炎症过程的重要参与者,其在慢性炎症的心力衰竭病人中水平显著升高[31]㊂相关研究显示,与健康对照组相比,DCM病人心肌细胞中的巨噬细胞数量无显著变化[32-33]㊂CD163是M2型巨噬细胞的标志,其在DCM中的研究结果存在争议㊂有研究报道,CD163阳性细胞数量的增加与DCM病人较差的预后显著相关,是心肌组织中胶原含量的独立预测因素,可能参与DCM的心室重塑[34]㊂有研究显示,CD163水平与DCM的严重程度无关[35]㊂本研究分析发现CD68和CD163的水平在PIDC病人中均是降低的㊂这些结果提示巨噬细胞可能通过复杂的机制参与PIDC的发生发展,仍需进一步研究㊂FCER1G作为固有免疫基因,参与炎症通路,诱导多种疾病发生[36]㊂一项体细胞和单细胞RNA测序数据的综合分析发现,FCER1G参与DCM的免疫过程[37],其与DCM或心力衰竭的关系,在这2种疾病病理生理状态中的作用仍待进一步研究㊂CCND1是重要的细胞周期调节蛋白,促进细胞周期从DNA合成前期(G1期)到DNA复制期(S期)的转换,其水平的上调参与多种肿瘤的进展[38]㊂基因肌联蛋白(TTN)的功能是编码肌节中最大的结构蛋白,其截段突变导致TTN缺乏是家族性和散发性DCM的主要原因㊂Liao等[39]研究显示,CCND1上调可抑制TTN缺乏引起的DCM,结果提示TTN不足导致DCM促进心肌细胞的细胞周期治疗,CCND1是其中一个重要的治疗靶点㊂核心基因在PIDC的发病机制仍待研究,可能成为治疗PIDC和DCM的新靶点㊂最后,本研究进行免疫细胞浸润分析发现,与对照组比较,试验组6种细胞含量有显著差异㊂CD8+T细胞在PIDC的心肌组织中含量升高㊂Luppi等[40-41]研究显示,DCM病人心肌组织中有CD8+T淋巴细胞浸润㊂Rao等[42]进一步研究提示,增多的CD8+T细胞主要位于纤维化的心肌中㊂上述研究结果提示CD8+T 细胞在PIDC的发病机制,特别在心室重构中发挥着重要的致病作用㊂DCM和心力衰竭病人中树突样细胞减少[32,43]㊂本研究分析发现树突样细胞在PIDC的心肌组织中含量降低㊂关于骨髓来源的抑制性细胞与DCM的研究较少,Zhang等[44]研究显示,DCM病人循环中数量显著升高,且与N末端脑钠肽前体呈正相关,与左室射血分数呈负相关㊂本研究分析发现,骨髓来源的抑制性细胞在PIDC病人心肌组织中减少,不一致的研究结果提示其在DCM发生发展中的作用有待进一步研究㊂本研究发现,PIDC心肌组织中自然杀伤T细胞含量减少㊂有研究显示,自然杀伤T细胞在DCM中含量降低[45]㊂已有基础研究显示,DCM小鼠脾脏中自然杀伤T细胞数量减少,补充外源性的树突样细胞可激活体内的自然杀伤T细胞,延长DCM小鼠生存,预防心脏收缩功能下降,抑制间质纤维化[46]㊂这些结果提示免疫细胞调节治疗可能成为治疗DCM 的新策略㊂综上所述,炎症及免疫反应在PIDC的发病机制中发挥着重要作用,5个核心基因和6种免疫细胞与PIDC发生发展密切相关,是其防治的潜在靶点㊂本研究对PIDC关键基因及信号通路进行了分子机制探讨,并辅以免疫细胞浸润分析,后期仍需进一步深入探讨免疫浸润细胞与关键基因或通路的关联性㊂参考文献:[1]PONZONI M,CASTALDI B,PADALINO M A.Pulmonary arterybanding for dilated cardiomyopathy in children:returning to thebench from bedside[J].Children,2022,9(9):1392.[2]WANG M,XU Y,WANG S,et al.Predictive value ofelectrocardiographic markers in children with dilatedcardiomyopathy[J].Frontiers in Pediatrics,2022,10:917730. 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