传染性胃肠炎病毒感染PK-15细胞环状RNA表达谱分析  

circRNA Expression Profiling Analysis of Transmissible Gastroenteritis Virus-infected PK-15 Cells

作　　者：杨玺望 杜芸莎 刘瑞 梅彩秋 李文庭 刘骁 YANG Xiwang;DU Yunsha;LIU Rui;MEI Caiqiu;LI Wenting;LIU Xiao(College of Veterinary Medicine,Southwest University,Chongqing 400715,China;The Second Affiliated Hospital of Hainan Medical University,Haikou 570100,China;Ya’an People’s Hospital,Ya’an 625000,China;State Key Laboratory of Silkworm Genome Biology,Southwest University,Chongqing 400715,China)

机构地区：[1]西南大学动物医学院,重庆400715 [2]海南医学院第二附属医院,海口570100 [3]雅安市人民医院,雅安625000 [4]西南大学,家蚕基因组生物学国家重点实验室,重庆400715

出　　处：《中国畜牧兽医》2024年第10期4450-4464,共15页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金项目(82260125);重庆市基础研究与前沿技术计划项目(cstc2020jcyj-msxm1458);四川省科技计划(2022YFN0069、2022YFQ0023、2021ZDZX0010);海南省自然科学基金项目(822MS181、823RC591);安徽省重点项目(S202104j07020097)。

摘　　要：【目的】分析传染性胃肠炎病毒(Transmissible gastroenteritis virus,TGEV)感染对猪肾上皮细胞(PK-15)中环状RNA(circRNA)表达的影响以及差异表达circRNA的潜在调控功能。【方法】利用Illumina HiSeq 4000测序平台对PK-15细胞(对照)和TGEV感染的PK-15细胞进行circRNA转录组测序,筛选差异表达circRNA。对差异表达circRNA来源基因进行GO功能和KEGG通路富集分析,预测TGEV感染相关circRNA-miRNA-mRNA调控网络。随机挑选12种差异表达circRNAs通过实时荧光定量PCR验证其表达水平。【结果】转录组测序中共发现1029种circRNAs。相较于对照PK-15细胞,TGEV感染的PK-15细胞中共发现128种显著差异表达circRNAs,其中70种上调,58种下调。GO功能分析显示,TGEV感染的PK-15细胞中差异表达circRNAs来源基因主要富集在细胞大分子代谢过程和细胞代谢等生物过程;细胞内部分和胞内膜结合细胞器等细胞成分;有机环化合物结合和核酸结合等分子功能中。KEGG通路富集结果显示,TGEV感染的PK-15细胞中差异表达circRNAs来源基因主要富集在肌动蛋白细胞骨架调控、甲型流感、丙型肝炎、cAMP信号通路和mTOR信号通路等方面。circRNA-miRNA-mRNA调控网络显示,TGEV感染的PK-15细胞中存在庞大复杂的circRNA调控网络,多种circRNAs可通过miRNA靶向基因IFNAR1、IFNAR2和NHE3来调节先天免疫和跨膜离子转运,从而影响TGEV感染和症状。实时荧光定量PCR鉴定结果显示,12种差异表达circRNAs表达水平与测序结果基本趋势一致。【结论】TGEV改变了PK-15细胞中circRNA的表达。差异表达circRNA来源基因广泛参与细胞大分子代谢、病毒感染及cAMP信号通路等过程,与先天性免疫和TGEV发病机制相关的多个靶基因IFNAR1、IFNAR2和NHE3可能被circRNA调控。本研究结果揭示了circRNA在TGEV与宿主相互作用中的潜在功能。【Objective】This study was aimed to analyze the effects of Transmissible gastroenteritis virus(TGEV)infection on circular RNA(circRNA)expression in porcine kidney epithelial cells(PK-15)and the potential regulatory functions of differentially expressed circRNA.【Method】Illumina HiSeq 4000 sequencing platform was used to sequence circRNA transcriptome of PK-15 cells(control)and TGEV-infected PK-15 cells,then screened differentially expressed circRNA.GO function and KEGG pathway enrichment analysis of differentially expressed circRNAs-derived genes were performed.TGEV infection-associated circRNA-miRNA-mRNA regulatory network was predicted.Moreover,12 differentially expressed circRNAs were randomly selected to verify their expression by Real-time quantitative PCR.【Result】Sequencing results revealed 1029 novel circRNAs.Compared with PK-15 cells,a total of 128 significantly differentially expressed circRNAs were identified in TGEV-infected PK-15 cells,of which 70 circRNAs were upregulated and 58 circRNAs were downregulated.GO functional analysis showed that the genes originating from differentially expressed circRNAs in TGEV-infected PK-15 cells were mainly enriched in biological processes such as cellular macromolecular metabolic processes and cellular metabolism,cellular components such as intracellular fractions and intracellular membrane-bound organelles,and molecular functions such as organic ring compound binding and nucleic acid binding.KEGG pathway enrichment results showed that the differentially expressed circRNAs source genes in TGEV-infected PK-15 cells were mainly enriched in actin cytoskeleton regulation,influenza A,hepatitis C,cAMP signaling pathway,mTOR signaling pathway,etc.The circRNA-miRNA-mRNA regulatory network revealed that there was a large and complex circRNA regulatory network in TGEV-infected PK-15 cells,and a variety of circRNAs regulated innate immunity and transmembrane ion transport by targeting genes IFNAR 1,IFNAR 2 and NHE 3 via miRNAs,thereby affecting TGEV infection and sym

关 键 词：环状RNA(circRNA) 传染性胃肠炎病毒(TGEV) PK-15细胞 转录组 

分 类 号：S852.659.6[农业科学—基础兽医学]