传染性胃肠炎病毒感染PK-15细胞环状RNA表达谱分析

doi:10.16431/j.cnki.1671-7236.2024.10.026

摘要/Abstract

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1 029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。

关键词: 环状RNA(circRNA); 传染性胃肠炎病毒(TGEV); PK-15细胞; 转录组

Abstract: 【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 1 029 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 IFNAR1，IFNAR2 and NHE3 via miRNAs，thereby affecting TGEV infection and symptoms. Real-time quantitative PCR showed that the expression of 12 differentially expressed circRNAs were consistent with the basal trend of RNA-Seq results. 【Conclusion】 TGEV altered circRNA expression in PK-15 cells. Differentially expressed circRNA-derived genes were widely involved in cellular macromolecular metabolism，viral infection and cAMP signaling pathways. Multiple target genes IFNAR1，IFNAR2 and NHE3 associated with innate immunity and TGEV pathogenesis were regulated by circRNA. The results revealed the potential function of circRNA in TGEV-host interactions.

Key words: circular RNA (circRNA); Transmissible gastroenteritis virus (TGEV); PK-15 cells; transcriptome

中图分类号:

S852.65⁺9.6

杨玺望, 杜芸莎, 刘瑞, 梅彩秋, 李文庭, 刘骁. 传染性胃肠炎病毒感染PK-15细胞环状RNA表达谱分析[J]. 中国畜牧兽医, 2024, 51(10): 4450-4464.

YANG Xiwang, DU Yunsha, LIU Rui, MEI Caiqiu, LI Wenting, LIU Xiao. circRNA Expression Profiling Analysis of Transmissible Gastroenteritis Virus-infected PK-15 Cells[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(10): 4450-4464.

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