禽腺病毒血清4型感染LMH细胞的环状RNA表达分析

doi:10.7668/hbnxb.20195576

华北农学报 ›› 2025, Vol. 40 ›› Issue (4): 219-224. doi: 10.7668/hbnxb.20195576

所属专题： 植物保护；畜牧；

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禽腺病毒血清4型感染LMH细胞的环状RNA表达分析

殷冬冬¹^,², 朱梦琪³, 兰梦蝶⁴, 朱星星³, 尹磊¹^,², 沈学怀¹^,², 王洁茹¹^,², 赵瑞宏¹^,², 戴银¹^,², 潘孝成¹^,²^,

¹ 安徽省农业科学院畜牧兽医研究所,安徽省畜禽疫病研究中心,安徽合肥 230001
² 安徽省畜禽产品安全重点实验室, 安徽合肥 230001
³ 安徽农业大学动物医学院,安徽合肥 231200
⁴ 宁国市动物卫生监督所,安徽宁国 242300

收稿日期:2024-12-05 出版日期:2025-08-28
通讯作者:
潘孝成(1971-),男,安徽霍邱人,副研究员,博士,主要从事畜禽病原微生物致病机制研究。
作者简介:
殷冬冬(1990-),男,安徽滁州人,助理研究员,博士,主要从事畜禽传染病防控研究。
基金资助:
财政部和农业农村部:国家现代农业产业技术体系(CARS-40); 安徽省农业科学院科研平台项目(2024YL016); 安徽省现代农业产业技术体系(AHCYJXTX-05-13)

Expression Analysis of circRNA in LMH Cells Infected with Fowl Adenovirus Serotype 4

YIN Dongdong¹^,², ZHU Mengqi³, LAN Mengdie⁴, ZHU Xingxing³, YIN Lei¹^,², SHEN Xuehuai¹^,², WANG Jieru¹^,², ZHAO Ruihong¹^,², DAI Yin¹^,², PAN Xiaocheng¹^,²^,

¹ Institute of Animal Husbandry and Veterinary Science,Anhui Academy of Agricultural Sciences, Livestock and Poultry Epidemic Diseases Research Center of Anhui Province,Hefei 230001,China
² Anhui Provincial Key Laboratory of Livestock and Poultry Product Safety,Hefei 230001,China
³ College of Veterinary Medicine,Anhui Agricultural University,Hefei 231200,China
⁴ Ningguo City Animal Health Supervision Institute,Ningguo 242300,China

Received:2024-12-05 Published:2025-08-28

摘要/Abstract

摘要：

为分析禽腺病毒血清4型(FAdV-4)感染鸡肝癌细胞(LMH)后环状RNA(circRNA)的表达谱变化,进一步研究circRNA在FAdV-4感染过程中的调控作用,以FAdV-4感染的LMH和未感染细胞为对象进行转录组测序,对差异表达circRNA进行GO功能和KEGG信号通路富集分析,通过实时荧光定量 PCR(qRT-PCR)对随机挑选的5个circRNA进行验证。结果表明,感染组和未感染组的circRNA分布于绝大多数染色体上,且长度主要集中在300~1 000 bp。差异表达分析筛选出72个circRNA,32个circRNA表达水平显著上调,40个circRNA表达水平下调。GO功能分析,差异circRNA来源基因主要富集在细胞过程、代谢过程、催化活性和核酸结合转录因子活性等进程。KEGG通路分析显示,差异表达circRNA主要富集在Notch信号通路、RNA降解和MAPK信号通路。qRT-PCR结果表明,被验证的5个circRNA表达水平与测序结果趋势一致,进一步验证了测序结果的可靠性。本研究对FAdV-4感染LMH细胞的circRNA表达谱进行分析,并筛选出差异表达的circRNA,为探究circRNA在FAdV-4感染过程中的功能及宿主与FAdV-4互作机制提供数据支持。

关键词: 禽腺病毒血清4型, LMH细胞, 转录组测序, 环状RNA, 差异表达

Abstract:

In order to analyze the alterations in the expression profile of circular RNA(circRNA)in chicken liver cancer cells line(LMH)infected by fowl adenovirus serotype 4(FAdV-4),and the regulatory role of circRNA in the FAdV-4 infecting process,transcriptome sequencing was carried out on FAdV-4-infected LMH cells and uninfected ones.Enrichment analysis of GO functions and KEGG signaling pathways was executed for differentially expressed circRNAs,and five randomly selected circRNAs were verified by Real-time Fluorescent Quantitative PCR(qRT-PCR).The results demonstrated that the circRNAs in the infected and uninfected groups were distributed on the preponderance of chromosomes,and their lengths were mainly concentrated between 300 and 1 000 bp.Differential expression analysis identified 72 circRNAs,with 32 showing significantly upregulated expression levels and 40 presenting downregulated expression levels.GO functional analysis revealed that the genes from which the differential circRNAs originated were mainly enriched in processes such as cellular processes,metabolic processes,catalytic activity,and nucleic acid-binding transcription factor activity.KEGG pathway analysis indicated that the differentially expressed circRNAs were primarily enriched in the Notch signaling pathway,RNA degradation,and the MAPK signaling pathway.The qRT-PCR results showed that the expression levels of the five verified circRNAs were consistent with the sequencing results,further validating the reliability of the sequencing results.This study analyzed the expression profile of circRNAs in FAdV-4-infected LMH cells and screened out differentially expressed circRNAs,providing data support for exploring the functions of circRNAs during the FAdV-4 infection process and the interaction mechanism between the host and FAdV-4.

Key words: Fowl adenovirus serotype 4, LMH cells, Transcriptome sequencing, Circular RNA, Differential expression

中图分类号:

S852.65

殷冬冬, 朱梦琪, 兰梦蝶, 朱星星, 尹磊, 沈学怀, 王洁茹, 赵瑞宏, 戴银, 潘孝成. 禽腺病毒血清4型感染LMH细胞的环状RNA表达分析[J]. 华北农学报, 2025, 40(4): 219-224. doi: 10.7668/hbnxb.20195576.

YIN Dongdong, ZHU Mengqi, LAN Mengdie, ZHU Xingxing, YIN Lei, SHEN Xuehuai, WANG Jieru, ZHAO Ruihong, DAI Yin, PAN Xiaocheng. Expression Analysis of circRNA in LMH Cells Infected with Fowl Adenovirus Serotype 4[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(4): 219-224. doi: 10.7668/hbnxb.20195576.

http://www.hbnxb.net/CN/Y2025/V40/I4/219

图/表 8

表1 qRT-PCR检测引物

Tab.1 Primers for qRT-PCR

引物名称 Primer name	引物序列(5'-3') Primer sequence (5'-3')
novel_circ_000130-F	GAAACCCCCAGCTACACGAA
novel_circ_000130-R	GCTCTCTGCCTTCAGGTTGT
novel_circ_000142-F	AGGAATGGAGAGATGCCCCT
novel_circ_000142-R	CAGTGCAGCTCCTTTCCAGA
novel_circ_000176-F	AGGATGAACGTCTGCACCAG
novel_circ_000176-R	GGAGAAGCCAACTGTCTGCT
novel_circ_000222-F	TGCAGAAAGCTGGGAAGGAG
novel_circ_000222-R	GCCTTTGGCTTCCTGATCCT
novel_circ_000261-F	CACCGCACTTAAGAGGCAGA
novel_circ_000261-R	TGACAGCCCCAACATGTACC
GAPDH-F	CAGAACATCATCCCAGCGTC
GAPDH -R	GGCAGGTCAGGTCAACAAC

表2 测序数据质量统计

Tab.2 Statistics of sequencing data quality

样品 Sample	原始序列/ 条 Raw reads	高质量序列/条 Clean reads	低质量序列/条 Low quality reads	质量值30/ % Q30
N1	97 103 488	96 902 158	183 408	93.47
N2	86 571 570	86 370 954	184 554	93.20
N3	91 907 490	91 686 366	203 302	93.28
T24-1	112 036 796	111 767 822	248 522	93.20
T24-2	117 365 326	117 092 796	256 164	93.22
T24-3	99 043 618	98 810 882	217 636	93.17

图1 circRNA的鉴定及特征 A.circRNA在FAdV-4感染组和未感染组LMH细胞中的染色体分布;B.circRNA的长度统计;C.circRNA的反向剪接读段统计。

Fig.1 Identification and characterization of circRNA A.Distribution of circRNA on chromosome in FAdV-4-infected and uninfected LMH cells; B.Length statistics of circRNA;C.Backspliced reads statistics of circRNA.

图2 差异表达circRNA的分析 A.差异表达circRNA柱状图;B.差异表达circRNA表达量聚类热图。

Fig.2 Differential expression analysis of circRNA A.Histogram of differentially expressed circRNA;B.Cluster heatmap of the expression levels of differential expression circRNA.

表3 部分差异表达的circRNA来源信息

Tab.3 Source information of some differentially expressed circRNA

环状RNA circRNA	差异倍数 log₂(FL)	来源基因 Source gene	染色体 Chromosomes	长度/bp Length
novel_circ_000007	-0.88	ncbi_424438	NC_006095.5	1 743
novel_circ_000039	-20.51	ncbi_423055	NC_00609.5	453
novel_circ_000057	-1.79	ncbi_39105245	NC_04090.1	272
novel_circ_000060	-0.42	ncbi_421086	NC_006089.5	1 809
novel_circ_000110	-20.09	ncbi_396504	NC_006106.5	780
novel_circ_000154	-19.98	ncbi_421002	NC_006089.5	1 347
novel_circ_000193	-20.15	ncbi_416092	NC_006099.5	593
novel_circ_000212	-19.99	ncbi_769652	NC_006088.5	889
novel_circ_000224	-20.46	ncbi_39105245	NC_04090.1	198

图3 差异表达circRNA来源基因GO富集分析 1.细胞过程;2.代谢过程;3.生物调节;4.生物过程调控;5.单一生物过程;6.应激反应;7.细胞成分组织或生物发生;8.多细胞生物过程;9.积极调节生物过程;10.发展过程;11.本地化;12.生物过程的负调控;13.信号;14.免疫系统过程;15.多生物过程;16.生物黏附;17.运动;18.生殖;19.生殖过程;20.行为;21.细胞杀伤;22.细胞;23.细胞区域;24.细胞器;25.薄膜;26.高分子复合物;27.膜部件;28.细胞器部分;29.膜封闭管腔;30.细胞连接;31.细胞外基质;32.细胞外区;33.细胞外区部分;34.结合;35.催化活性;36.核酸结合转录因子活性;37.分子功能调节;38.转运活性;39.分子转导活性;40.信号传导活性;41.蛋白质结合。

Fig.3 GO functional enrichment analysis of target genes of differentially expressed circRNA 1.Cellular process;2.Metabolic process;3.Biological regulation;4.Regulation of biological process;5.Single-organism process;6.Response to stimulus;7.Cellular component organization or biogenesis;8.Multicellular organismal process;9.Positive regulation of biological process;10.Developmental process;11.Localization;12.Negative regulation of biological process;13.Signaling;14.Immune system process;15.Multi-organism process;16.Biological adhesion;17.Locomotion;18.Reproduction;19.Reproductive process;20.Behavior;21.Cell killing;22.Cell;23.Cell part;24.Organelle;25.Membrane;26.Macromolecular complex;27.Membrane part;28.Organelle part;29.Membrane-enclosed lumen;30.Cell junction;31.Extracellular matrix;32.Extracellular region;33.Extracellular region part;34.Binding;35.Catalytic activity;36.Nucleic acid binding transcription factor activity;37.Molecular function regulator;38.Transporter activity;39.Molecular transducer activity;40.Signal transducer activity;41.Protein binding.

图4 差异表达circRNA来源基因KEGG通路富集分析

Fig.4 KEGG pathway enrichment analysis of target genes of differentially expressed circRNA

图5 qRT-PCR验证差异表达circRNA

Fig.5 Validation of differentially expressed circRNA by qRT-PCR

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禽腺病毒血清4型感染LMH细胞的环状RNA表达分析

Expression Analysis of circRNA in LMH Cells Infected with Fowl Adenovirus Serotype 4

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