Liu Haiyang, Liu Di, Liu Yexin, Xia Ming, Li Yan, Li Mei, Liu Hong
Department of Nephrology, The Second Xiangya Hospital of Central University, Hunan Key Laboratory of Kidney Disease and Blood Purification, Changsha, Hunan, China.
PeerJ. 2020 Dec 3;8:e10395. doi: 10.7717/peerj.10395. eCollection 2020.
Immunoglobulin A nephropathy (IgAN) is immune-mediated primary glomerulonephritis, which is the most common reason leading to renal failure worldwide. The exact pathogenesis of IgAN is not well defined. Accumulating evidence indicates that circular RNAs (circRNAs) play crucial roles in the immune disease by involving in the competing endogenous RNA (ceRNA) network mechanism. At present, the studies of the circRNA profiles and circRNA-associated ceRNA networks in the IgAN are still scarce. This study aimed to elucidate the potential roles of circRNA-associated ceRNA networks of peripheral blood mononuclear cells (PBMCs) in IgAN patients.
CircRNA sequencing was used to identify the differential expressed circRNAs (DEcircRNAs) of PBMCs in IgAN and healthy controls; limma packages from data sets GSE25590 and GSE73953 in the Gene Expression Omnibus (GEO) database, were used to identify differentially expressed micro RNAs (miRNAs) and message RNAs (mRNAs). A circRNA-miRNA-mRNA ceRNA network was constructed to further investigate the mechanisms of IgAN. Then, GO analysis and KEGG enrichment analyses were used to annotate the genes involved in the circRNA-associated ceRNA network. Further, Protein-protein interaction (PPI) networks were established to screen potential hub genes, by using Search Tool for the Retrieval of Interacting Genes/Proteins (STRING). Last, a quantitative real-time polymerase chain reaction (qRT-PCR) was applied to verify the hub genes in the ceRNA network.
A total of 145 circRNAs, 22 miRNAs, and 1,117 mRNAs were differentially expressed in IgAN compared with controls ( < 0.05). A ceRNA network was constructed which contained 16 DEcircRNAs, 72 differential expressed mRNAs (DEmRNAs) and 11 differential expressed miRNAs (DEmiRNAs). KEGG pathway enrichment analysis illustrated the underlying biological functions of the ceRNA-associated genes, such as Nitrogen compound metabolic process, COPII-coated ER to Golgi transport vesicle, CAMP response element protein binding process ( < 0.01); meanwhile, Hepatitis B, GnRH signaling, and Prion disease were the most significant enrichment GO terms ( < 0.01). PPI network based on STRING analysis identified 4 potentially hub genes. Finally, Ankyrin repeat and SOCS box containing 16 (ASB16), SEC24 homolog C, COPII coat complex component (SEC24C) were confirmed by qRT-PCR ( < 0.05) and were identified as the hub genes of the ceRNA network in our study.
Our study identified a novel circRNA-mediated ceRNA regulatory network mechanisms in the pathogenesis of IgAN.
免疫球蛋白A肾病（IgAN）是一种免疫介导的原发性肾小球肾炎，是全球范围内导致肾衰竭的最常见原因。IgAN的确切发病机制尚不明确。越来越多的证据表明，环状RNA（circRNA）通过参与竞争性内源RNA（ceRNA）网络机制在免疫疾病中发挥关键作用。目前，关于IgAN中circRNA谱和circRNA相关ceRNA网络的研究仍然很少。本研究旨在阐明IgAN患者外周血单个核细胞（PBMC）中circRNA相关ceRNA网络的潜在作用。
采用circRNA测序鉴定IgAN患者和健康对照者PBMC中差异表达的circRNA（DEcircRNA）；使用基因表达综合数据库（GEO）中数据集GSE25590和GSE73953的limma软件包，鉴定差异表达的微小RNA（miRNA）和信使RNA（mRNA）。构建circRNA-miRNA-mRNA ceRNA网络以进一步研究IgAN的发病机制。然后，使用GO分析和KEGG富集分析对circRNA相关ceRNA网络中涉及的基因进行注释。此外，通过使用搜索相互作用基因/蛋白质的工具（STRING）建立蛋白质-蛋白质相互作用（PPI）网络，以筛选潜在的枢纽基因。最后，应用定量实时聚合酶链反应（qRT-PCR）验证ceRNA网络中的枢纽基因。
与对照组相比，IgAN中共有145个circRNA、22个miRNA和1117个mRNA差异表达（P<0.05）。构建了一个ceRNA网络，其中包含16个DEcircRNA、72个差异表达的mRNA（DEmRNA）和11个差异表达的miRNA（DEmiRNA）。KEGG通路富集分析阐明了ceRNA相关基因的潜在生物学功能，如氮化合物代谢过程、COPII包被的内质网到高尔基体运输囊泡、CAMP反应元件蛋白结合过程（P<0.01）；同时，乙型肝炎、GnRH信号传导和朊病毒病是最显著的富集GO术语（P<0.01）。基于STRING分析的PPI网络鉴定出4个潜在的枢纽基因。最后，通过qRT-PCR验证了锚蛋白重复序列和含SOCS盒蛋白16（ASB16）、SEC24同源物C、COPII包被复合物成分（SEC24C）（P<0.05），并将其鉴定为本研究中ceRNA网络的枢纽基因。
我们的研究在IgAN发病机制中鉴定出一种新的circRNA介导的ceRNA调控网络机制。
