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

doi:10.7668/hbnxb.20195576

Abstract

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

CLC Number:

S852.65

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.

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

Figures/Tables 8

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

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

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.

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

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

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.

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

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

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