H1N1亚型流感病毒感染A549细胞的环状RNA表达分析

doi:10.11843/j.issn.0366-6964.2023.11.026

摘要/Abstract

摘要： 通过对甲型H1N1流感病毒感染A549细胞前后表达的环状RNA进行鉴定,筛选病毒感染引起的差异表达环状RNA,为进一步研究环状RNA在流感病毒感染过程中的调控作用奠定基础。本研究从病毒感染和PBS处理的A549细胞中提取RNA,构建测序文库后进行全转录组测序。通过生信分析,鉴定了病毒感染前后宿主细胞表达的环状RNA。以未感染组为对照,使用EdgeR包,筛选条件:log₂FC≥1、P value<0.05,筛选获得感染前后的细胞中差异表达的环状RNA。使用ClusterProfiler包对差异表达环状RNA的来源基因进行GO和KEGG分析。最后通过荧光定量PCR、外切核糖核酸酶处理、Sanger测序等手段验证了病毒感染前后宿主细胞内部分环状RNA的差异表达。结果表明:流感病毒感染细胞后,环状RNA的表达数目增加。感染组和未感染组分别鉴定到1 101和676个环状RNA表达,这些环状RNA广泛分布在所有染色体上,其中大部分(约60%)长度在300~1 000 nt,约20%的环状RNA长度超过2 000 nt。基于基因位置关系的分析发现,75%~82%的环状RNA源自外显子,12%~17%源自正链重叠转录本,少部分(1%~4%)来自内含子、基因间和反义链转录本。经差异表达分析,共筛选出54个表达上调,18个表达下调的环状RNA。这些差异表达的环状RNA主要分布在5号染色体和19号染色体上。基因本体论GO富集分析显示,差异表达环状RNA来源基因主要富集到RNA定位、细胞质膜和单链DNA结合等相关进程。KEGG通路分析显示,差异表达环状RNA主要与氨基酸降解、FoxO信号等通路相关。最后,通过RT-qPCR、RNase R消化、Sanger测序等方法,验证了环状RNA SNUPN(has_circ_0104558)、DCBLD1(hsa_circ_0077717)和EIF4G3(hsa_circ_0000025)在病毒感染前后细胞中的差异表达,证实其是由对应来源序列反向剪接而成并且具有环状RNA抗RNase R降解的特性。本研究通过转录组测序和生信分析手段鉴定了H1N1甲型流感病毒感染A549细胞前后环状RNA的表达谱,分析了环状RNA的长度、染色体分布、来源分类和可能参与的调控通路,筛选出了病毒感染后差异表达的环状RNA,为深入研究环状RNA是否在流感病毒感染期间发挥作用奠定了基础。

关键词: 环状RNA, 甲型流感病毒, A549细胞, 差异表达

Abstract: The objective of this study was to identify the differentially expressed circRNAs in A549 cells upon influenza A virus infection. The findings will provide a basis for future researches on the regulatory roles of circular RNAs (circRNAs) in influenza A virus replication. RNA extracted from IAV-infected A549 cells and PBS-treated control cells were subjected to whole transcriptome sequencing. Bioinformatic analysis was performed to identify the circRNAs expressed in the cells. Comparing with uninfected control samples, differentially expressed circRNAs in cells before and after infection were screened using the EdgeR package with the selection criteria of log₂FC≥1 and P value<0.05. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted on the parental genes of the differentially expressed circRNAs using the ClusterProfiler package. Finally, the differential expression of some circRNAs before and after viral infection was verified by fluorescence quantitative PCR, exonuclease treatment, and Sanger sequencing. The abundance and varity of circRNAs were found to increase upon influenza virus infection. By RNA sequencing, a total of 1 101 and 676 circRNAs were identified in the infected and uninfected groups, respectively. These circRNAs were widely distributed across all chromosomes, with approximately 60% ranging in length from 300 nt to 1 000 nt, and around 20% of circRNAs being longer than 2 000 nt. Furthermore, 75%-82% of circRNAs were derived from exons, while 12%-17% were classified as sense overlapping, and a small fraction (1%-4%) originated from intronic, intergenic, or antisense transcripts. Differential expression analysis identified 54 up-regulated and 18 down-regulated circRNAs in response to viral infection. These differentially expressed circRNAs were mainly distributed on chromosomes 5 and 19. Gene ontology (GO) enrichment analysis revealed that the parent genes of differentially expressed circRNAs were significantly enriched in RNA localization, cytoplasmic membrane, and single-stranded DNA binding. Additionally, KEGG pathway analysis demonstrated that these circRNAs were mainly enriched in pathways related to amino acid degradation and FoxO signaling. Finally, the differential expression of three circRNAs (SNUPN:has_circ_0104558, DCBLD1:hsa_circ_0077717, and EIF4G3:hsa_circ_0000025) was verified by RT-qPCR, RNase R digestion, and Sanger sequencing, which confirmed that they were back spliced from the corresponding source sequences and were resistant to RNase R digestion. The circRNA expression profiles in A549 cells upon influenza A virus infection were obtained using transcriptome sequencing and bioinformatics analysis. The length, chromosome distribution, source classification of these circRNAs, and the potential pathways they probably involved in were analyzed. We identified differentially expressed circRNAs upon viral infection, which lays a foundation for further investigations on the potential roles of circRNAs in influenza virus infection.

Key words: circRNA, influenza A virus, A549 cells, differential expression

中图分类号:

S852.659.5

蒋盛强, 胡靖, 陈红英. H1N1亚型流感病毒感染A549细胞的环状RNA表达分析[J]. 畜牧兽医学报, 2023, 54(11): 4724-4734.

JIANG Shengqiang, HU Jing, CHEN Hongying. Expression Analysis of CircRNAs in A549 Cells Infected with H1N1 Influenza A Virus[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(11): 4724-4734.

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