基于ChIP-seq技术分析心肌成纤维细胞中KDM5A调控的下游靶基因网络

Investigation of the downstream target gene network regulated by KDM5A

目的 探讨KDM5A调控心肌成纤维细胞生物学功能的潜在机制。方法 获取2019年1月—2020年3月于蚌埠医学院第一附属医院心脏外科就诊的扩张型心肌病患者的心室肌组织。分离培养心肌纤维化患者的心肌成纤维细胞,采用染色质免疫共沉降测序(ChIP-seq)对细胞中KDM5A富集的DNA序列进行分析,对筛选出的差异基因进行基因本体论(GO)分析和京都基因和基因组百科全书(KEGG)通路富集分析。结果 KDM5A的结合峰在基因启动子区、内含子区和外显子区所占的比例分别为14.59%、44.14%和1.84%。其中,≤1 kb、>1~2 kb和>2~3 kb的启动子区比例分别为5.91%、4.56%和4.12%。KDM5A结合在第一内含子区和其他内含子区的比例分别为12.19%和31.95%。KDM5A结合峰相对于转录起始位点(TSS)的分布主要集中在-3~3 kb,在TSS坐标-1.0 kb和2.5 kb处的reads计数频率最大。利用Homer软件分别识别出KDM5A结合的motif保守序列包括FOXE1、PB、ZNF460等20个。GO功能注释分析提示,与KDM5A结合的基因涉及细胞形态发生的调控、轴突发育、神经元投射发育的调节、细胞质膜蛋白复合物、阳离子通道、肌动蛋白骨架、金属离子跨膜转运蛋白活性、阳离子跨膜转运蛋白活性、GTP酶结合等。KEGG信号通路富集结果显示,KDM5A相关基因显著富集在磷脂酶D信号通路、胆碱能突触、钙信号通路、Wnt信号通路、生长激素的合成、分泌和作用、胰岛素分泌、ErbB信号通路、Rap1信号通路等通路。结论 KDM5A通过调节基因的转录表达,调控信号通路表达,进而影响心肌成纤维细胞功能,参与心肌纤维化的发生与发展。

Objective To investigate the potential mechanism of KDM5A in regulating the biological function of cardiac fibroblasts.Methods Cardiac fibroblasts from patients with myocardial fibrosis were isolated and cultured,chromatin immunoprecipitation sequence(ChIP-seq)was performed to analyze immunoaffinity separation of specific binding DNA fragments with KDM5A for sequence identification,differentially expressed genes(DEGs)were screened for gene ontology(GO)and Kyoto encyclopedia of genes and genomes(KECG)pathway analysis.Results The binding peak of KDM5A in the gene promoter region,intron region,and exon region accounted for 14.59%,44.14%and 1.84%,respectively.Among them,the proportion of≤1 kb,>1-2 kb and>2-3 kb promoter regions were 5.91%,4.56%and 4.12%,respectively.The binding ratio of KDM5A to the first intron region and other intron regions were 12.19%and 31.95%,respectively.The distribution of KDM5A binding peaks relative to the transcription start site(TSS)was mainly concentrated at-3 to 3 kb,and the reads count frequency was the highest at the TSS coordinates-1.0 kb and 2.5 kb.Homer sofware was applied to identify 20 motifs that bind to KDM5A,including FOXE1,PB,ZNF460,etc.GO functional annotation analysis suggested that the KDM5A binding genes were involved in the regulation of cell morphogenesis,axon development,regulation of neuron projection development,plasma membrane protein complex,cation channel,actin cytoskeleton,metal ion transmembrane transporter activity,cation transmembrane transporter activity and GTP enzyme binding,etc.The KEGG signaling pathway enrichment results showed that KDM5A-related genes were significantly enriched in the phospholipase D signaling pathway,cholinergic synapse,calcium signaling pathways,Wnt signaling pathway,growth hormone synthesis,secretion and action,insulin secretion,ErbB signaling pathway and Rapl signaling pathways,etc.Conclusion KDM5A regulates the transcription expression of genes and the expression of signal pathways,thereby affecting the function of cardiac fibroblasts and participating in the onset and development of myocardial fibrosis.