Table_9_Identification of Potential Immune-Related circRNA–miRNA–mRNA Regulatory Network in Intestine of Paralichthys olivaceus During Edwardsiella tarda Infection.xlsx

Olive flounder (Paralichthys olivaceus) is an important economical flatfish in Japan, Korea, and China, but its production has been greatly threatened by disease outbreaks. In this research, we aimed to explore the immune responsive mechanism of P. olivaceus against Edwardsiella tarda infection by profiling the expression of circRNA, miRNA, and mRNA by RNA-seq and constructing a regulatory circular circRNA–miRNA–mRNA network. Illumina sequencing of samples from normal control (H0), 2 h (H2), 8 h (H8), and 12 h (H12) post-challenge was conducted. Differentially expressed (DE) circRNA (DE–circRNAs), miRNAs (DE–miRNAs), and mRNAs [differential expression genes (DEGs)] between challenge and control groups were identified, resulting in a total of 62 DE–circRNAs, 39 DE–miRNAs, and 3,011 DEGs. Based on the differentially expressed gene results, miRNA target interactions (circRNA–miRNA pairs and miRNA–mRNA pairs) were predicted by MiRanda software. Once these paired were combined, a preliminary circRNA–miRNA–mRNA network was generated with 198 circRNA–miRNA edges and 3,873 miRNA–mRNA edges, including 44 DE–circRNAs, 32 DE–miRNAs, and 1,774 DEGs. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was performed to evaluate the function of the DEGs in this network, and we focused and identified two important intestinal immune pathways (herpes simplex infection and intestinal immune network for IgA production) that showed statistical significance between the challenge and control groups. Furthermore, three critical DEGs (nectin2, MHC II α-chain, and MHC II β-chain) were identified, mapped into the preliminary circRNA–miRNA–mRNA network, and new circRNA–miRNA–mRNA regulatory networks were constructed. In conclusion, we, for the first time, identified circRNA–miRNA–mRNA network from P. olivaceus in the pathogenesis of E. tarda and provided valuable resources for further analyses of the molecular mechanisms and signaling networks.

褐牙鲆（Paralichthys olivaceus）是中日韩地区重要的经济比目鱼，但其养殖生产已被疾病暴发严重威胁。本研究旨在通过RNA测序（RNA-seq）分析环状RNA（circRNA）、微小RNA（miRNA）及信使RNA（mRNA）的表达谱，探索褐牙鲆抗迟缓爱德华氏菌（Edwardsiella tarda）感染的免疫应答机制，并构建调控性circRNA–miRNA–mRNA网络。研究对感染后正常对照组（H0）、感染后2小时（H2）、8小时（H8）及12小时（H12）的样本进行了Illumina测序。鉴定感染组与对照组间的差异表达circRNA（DE–circRNAs）、差异表达miRNA（DE–miRNAs）及差异表达基因（differential expression genes, DEGs），最终共获得62个DE–circRNAs、39个DE–miRNAs及3011个DEGs。基于差异表达基因的分析结果，通过MiRanda软件预测miRNA靶标互作关系（circRNA–miRNA对及miRNA–mRNA对）。将上述配对组合后，构建得到包含198个circRNA–miRNA互作边与3873个miRNA–mRNA互作边的初步circRNA–miRNA–mRNA网络，涉及44个DE–circRNAs、32个DE–miRNAs及1774个DEGs。对该网络中的DEGs进行京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）富集分析，筛选出感染组与对照组间具有统计学显著性的两条重要肠道免疫通路——单纯疱疹感染通路及IgA生成肠道免疫网络通路。进一步鉴定出3个关键DEGs：nectin2、主要组织相容性复合体II α链（MHC II α-chain）及主要组织相容性复合体II β链（MHC II β-chain），将其映射至初步circRNA–miRNA–mRNA网络后，构建得到新的circRNA–miRNA–mRNA调控网络。综上，本研究首次在迟缓爱德华氏菌感染的褐牙鲆中鉴定出circRNA–miRNA–mRNA调控网络，为后续解析其分子机制及信号网络提供了宝贵的研究资源。