精神分裂症患者外周血中差异表达circRNA、miRNA、mRNA筛选及核心竞争性内源RNA网络构建

Screening of differentially expressed circRNAs,miRNAs,and mRNAs in peripheral blood of schizophrenia patients and construction of a core ceRNA network

目的筛选精神分裂症患者外周血中差异表达的circRNA、miRNA、mRNA,并构建核心竞争性内源RNA(ceRNA)网络,解析精神分裂症发生发展的分子机制。方法精神分裂症患者6例记为精神分裂症组,同期体检健康的人群4例记为健康对照组。采集两组研究对象的外周血,提取总RNA后构建cDNA文库并进行高通量测序,采用R软件筛选精神分裂症患者外周血中差异表达circRNA、miRNA、mRNA。采用miRanda和qTar数据库共同预测与差异表达miRNA相互作用的circRNA和mRNA,与差异表达的circRNA、mRNA取交集后,采用R软件构建差异表达的ceRNA网络。基于DAVID数据库对精神分裂症患者差异表达ceRNA网络中差异mRNA进行GO和KEGG富集分析。采用string数据库构建精神分裂症患者差异表达ceRNA网络中差异mRNA之间的PPI网络,选用cytoscape软件中cytoHubba插件筛选影响精神分裂症发生发展的核心mRNA,选择与核心mRNA存在相互作用关系的miRNA和circRNA构建精神分裂症患者外周血中差异表达的核心ceRNA网络。结果相比健康对照组,精神分裂症组患者外周血中差异表达circRNA、miRNA、mRNA分别为31、213、2624个。构建了由差异表达的5个circRNA、4个miRNA和57个mRNA组成的ceRNA网络。精神分裂症患者差异表达ceRNA网络中mRNA主要参与化学刺激的感官知觉、多细胞生物生长的负调控等生物过程,涉及泛素-泛素连接酶活性、鸟苷酸结合、酶结合等分子功能,以及细胞质、肌动蛋白细胞骨架、轴突末端等细胞成分,ceRNA网络中mRNA显著富集于NF-κB信号通路、PI3K-Akt信号通路、Ras信号通路、泛素介导的蛋白水解等代谢通路。构建的精神分裂症患者差异表达ceRNA网络中mRNA PPI网络共包含23个节点和22条边,筛选获得影响精神分裂症发生发展的2个核心mRNA为BRCA1、GNAS。构建的精神分裂症患者外周血中差异表达的核心ceRNA网络包含5个节点和4条边。5个节点中包含2个核心mRNA:BRCA1、GNAS,1个miRNA:hsa-miR-502-5p,2个circRNA:hsa_circ_0001423、hsa_circ_0008494。4条边分别为hsa_circ_0001423/hsa-miR-502-5p/BRCA1、hsa_circ_0001423/hsa-miR-502-5p/GNAS、hsa_circ_0008494/hsa-miR-502-5p/BRCA1和hsa_circ_0008494/hsa-miR-502-5p/GNAS。结论与健康对照人群相比,精神分裂症患者外周血中有差异表达的circRNA 31个、miRNA 213个、mRNA 2624个,最终构建了包含5个节点和4条边的核心ceRNA网络。

Objective To screen the differentially expressed circRNAs,miRNAs,and mRNAs in the peripheral blood of patients with schizophrenia,to construct a core competitive endogenous RNA(ceRNA)network,and to elucidate the molecular mechanism underlying the development and progression of schizophrenia.Methods Six patients with schizophrenia were included in the schizophrenia group,and four healthy individuals undergoing physical examinations during the same period were included in the healthy control group.Peripheral blood was collected from both groups,and total RNA was extracted to construct cDNA libraries for high-throughput sequencing.R software was used to screen for differentially expressed circRNAs,miRNAs,and mRNAs in the peripheral blood of patients with schizophrenia.The miRanda and qTar databases were jointly used to predict circRNA and mRNA interacting with the differentially expressed miRNAs.After intersecting with the differentially expressed circRNA and mRNA,R software was used to construct a network of differentially expressed ceRNA.The DAVID database was used to perform Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses on the differentially expressed mRNAs in the ceRNA network of patients with schizophrenia.The STRING database was used to construct a protein-protein interaction(PPI)network among the differentially expressed mRNAs in the ceRNA network of patients with schizophrenia.The cytoHubba plugin in Cytoscape software was used to screen the core mRNAs that influenced the occurrence and development of schizophrenia.MiRNAs and circRNAs interacting with the core mRNAs were selected to construct a core ceRNA network with differentially expressed genes in the peripheral blood of patients with schizophrenia.Results Compared with the healthy control group,31 circRNAs,213 miRNAs,and 2624 mRNAs were differentially expressed in the peripheral blood of the schizophrenia group.A ceRNA network composed of 5 differentially expressed circRNAs,4 miRNAs,and 57 mRNAs was constructed.The mRNAs in the ceRNA network of patients with schizophrenia were mainly involved in biological processes such as sensory perception of chemical stimuli and negative regulation of multicellular organism growth.They were associated with molecular functions such as ubiquitin-ubiquitin ligase activity,guanine nucleotide binding,and enzyme binding,as well as cellular components such as the cytoplasm,actin cytoskeleton,and axon terminus.The mRNAs in the ceRNA network were significantly enriched in metabolic pathways such as NF-κB signaling pathway,PI3K-Akt signaling pathway,Ras signaling pathway,and ubiquitin-mediated proteolysis.The PPI network of mRNAs in the differentially expressed ceRNA network of patients with schizophrenia included 23 nodes and 22 edges,and two core mRNAs,BRCA1 and GNAS,that influenced the occurrence and development of schizophrenia were screened out.The core ceRNA network with differentially expressed genes in the peripheral blood of patients with schizophrenia included 5 nodes and 4 edges.The 5 nodes comprised 2 core mRNAs(BRCA1 and GNAS),1 miRNA(hsa-miR-502-5p),and 2 circRNAs(hsa_circ_0001423 and hsa_circ_0008494).The 4 edges were hsa_circ_0001423/hsa-miR-502-5p/BRCA1,hsa_circ_0001423/hsa-miR-502-5p/GNAS,hsa_circ_0008494/hsa-miR-502-5p/BRCA1,and hsa_circ_0008494/hsa-miR-502-5p/GNAS,respectively.Conclu⁃sion Compared with healthy controls,31 circRNAs,213 miRNAs,and 2624 mRNAs were differentially expressed in the peripheral blood of patients with schizophrenia,and we constructed the core ceRNA network with 5 nodes and 4 edges.