Abstract
Introduction
Newcastle disease virus (NDV) is prevalent worldwide with an extensive
host range. Among birds infected with velogenic NDV strains, chickens
experience high pathogenicity and mortality, whereas ducks mostly
experience mild symptoms or are asymptomatic. Ducks have a unique,
innate immune system hypothesized to induce antiviral responses.
Circular RNAs (circRNAs) are among the most abundant and conserved
eukaryotic transcripts. These participate in innate immunity and host
antiviral response progression.
Methods
In this study, circRNA expression profile differences post-NDV
infection in duck embryo fibroblast (DEF) cells were analyzed using
circRNA transcriptome sequencing. Gene Ontology (GO) and Kyoto
Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were used
to reveal significant enrichment of differentially expressed (DE)
circRNAs. The circRNA-miRNA-mRNA interaction networks were used to
predict the related functions of circRNAs. Moreover, circ-FBXW7 was
selected to determine its effect on NDV infection in DEFs.
Results
NDV infection altered circRNA expression profiles in DEF cells, and 57
significantly differentially expressed circRNAs were identified
post-NDV infection. DEF responded to NDV by forming circRNAs to
regulate apoptosis-, cell growth-, and protein degradation-related
pathways via GO and KEGG enrichment analyses. circRNA-miRNA-mRNA
interaction networks demonstrated that DEF cells combat NDV infection
by regulating cellular pathways or apoptosis through circRNA-targeted
mRNAs and miRNAs. circ-FBXW7 overexpression and knockdown inhibited and
promoted viral replication, respectively. DEF cells mainly regulated
cell cycle alterations or altered cellular sensing to combat NDV
infection.
Conclusion
These results demonstrate that DEF cells exert antiviral responses by
forming circRNAs, providing novel insights into waterfowl antiviral
responses.
Keywords: waterfowl, Newcastle disease virus, duck embryo fibroblasts,
circular RNAs, antiviral response
1. Introduction
Newcastle disease (ND), caused by the ND virus (NDV), was first
identified in Indonesia in 1926 ([43]1). ND is the most prevalent
poultry infectious disease worldwide and causes severe economic losses
to poultry farming industries ([44]1). NDV infects various species,
including chickens, turkeys, wild birds, and waterfowl, and undergoes
cross-species transmission ([45]2, [46]3). Waterfowls are natural NDV
hosts or carriers but are resistant to virulent strains ([47]4, [48]5).
Chickens and ducks infected with the genotype VII virulent NDV strain,
widespread in Asia, have variable mortality, with viruses detectable in
duck organs ([49]6, [50]7). Waterfowls infected with NDV strains do not
exhibit overt clinical signs but can release the virus via the
oropharynx or cloaca ([51]8, [52]9). Ducks possess RIG-I, and with its
absence in chickens, different natural immune responses to resist viral
infection occur ([53]10, [54]11). However, ducks exhibit resistance to
NDV through unique, innate immunity, although the specific resistance
mechanism remains undefined.
Circular RNA (circRNA) is eukaryotic covalently closed noncoding RNAs
mainly derived from back splicing in the formation of mRNA ([55]12).
circRNAs possess a covalently closed loop without 5′ or 3′ polarities
and a poly(A) tail configuration, conferring greater stability than
linear RNA ([56]13). They were originally found in plant and hepatitis
viruses and considered splicing error products during mRNA formation
([57]14). With continuous advances in high-throughput sequencing and
RNA research, several circRNAs have been identified in diverse species
and cell lines, and various circRNAs libraries have been supplemented
([58]15–[59]18). circRNA formation is a common and abundant regulation
method for gene expression programs in various species; circRNAs are
hypothesized to be evolutionarily conserved and show general features
during gene expression ([60]19). According to their source gene,
circRNAs are classified as exonic, exon-intron, and intronic circRNAs
([61]20).
circRNAs exert their functions by suppressing miRNA expression by
functioning as specific miRNA “sponges,” acting as designated miRNA
“reservoirs” to stabilize miRNA functions, modulating gene expression,
and encoding proteins ([62]20). Moreover, circRNA vaccine expressing a
trimeric receptor-binding domain (RBD) of SARS-CoV-2 spike proteins
could elicit potent neutralizing antibody and T-cell responses,
conferring protection against SARS-CoV-2 in mice and rhesus monkeys
([63]21). circRNAs in exosomes have been determined; their expression
profiles in cancer patient sera differed from those of healthy
individuals, suggesting circRNAs as possible biomarkers for cancer
diagnosis ([64]22). circRNAs participate in antiviral immune responses
and viral infections affect the host circRNA expression profiles
([65]23, [66]24). circRNAs also participate in antiviral responses
against Ebola virus, simian virus 40 (SV40), and avian leukosis virus
([67]25–[68]27), indicating that circRNAs might participate in the host
antiviral response.
Although circRNA expression has been detected in multiple species and
cell lines, research on duck circRNAs is limited. Virulent NDV strains
can infect waterfowls and chickens; however, they have different
incidence and mortality rates ([69]7). Therefore, next-generation
genome sequencing was used in this study to determine and analyze
circRNAs expression profiles post-NDV infection in duck cells, and
enriched key circRNA functions were explored. Our results suggested
that ducks might exert antiviral responses through circRNAs, providing
a theoretical basis for studying waterfowl antiviral responses.
2. Materials and methods
2.1. Viruses and cells
The NDV strain rGM was preserved in our laboratory ([70]28), multiplied
in 9-day-old embryonated specific-pathogen-free (SPF) chicken eggs, and
stored at −80°C. Duck embryo fibroblasts (DEF) cells were cultured and
maintained in DMEM (GIBCO, Grand Island, NY, United States)
supplemented with 10% FBS. Duck embryo fibroblast (DEF) cells were
prepared from 9 to 10-day-old embryonated eggs as per previously
described chicken embryo fibroblast (CEF) cell preparation ([71]29).
All NDV infection related experiments were conducted in an animal
biosafety level 3 facility, the protocol (SCAUABSL2022-R003) was
approved by the Animal Welfare Ethics Committee of South China
Agricultural University.
2.2. Median tissue culture infectious dose
Determination of TCID[50] was conducted as previously described
([72]30). DEF cells were seeded on 96-well plates with 15,000 cells in
100 μL of media per well. Plates were incubated at 37°C with 5% CO[2].
The following day, the media was aspirated and replaced by 100 μL of
media containing a serial dilution of the virus (1:10–1:100). After
3 days of incubation, wells were analyzed on a standard light
microscope for cytopathic effect (CPE), consisting of rounded cells, a
disrupted monolayer, and clumps. The number of CPE-positive wells in
each column was used to quantify the experiment by the methods of
Reed-Muench ([73]31). Briefly, DEF cells were infected with the rGM
strain at a multiplicity of infection (MOI) of 1 in single cycle. The
infected cultures were harvested at 4, 9, 12, and 24 h post-infection
(hpi). The rGM viral titers were determined in triplicates with the
standard median tissue culture infective dose assay ([74]28).
2.3. Construction of circRNA library
Trizol was used to extract total RNA from DEF cells. Agarose gel
electrophoresis was used in testing the integrity of the samples.
Meanwhile, the A260/A280 ratio for the Nanodrop detection of RNA should
be in the range from 1.8 to 2.0. Following rRNA depletion with Illumina
Ribo Zero Gold Kit and liner RNA degradation with RNase R, circRNA was
broken into short sections. A complete cDNA was obtained by addition of
random hexamers, buffer, dNTPs, RNase H and DNA polymerase. To
establish a whole library, the system was first configured according to
the QiaQuick PCR kit protocol, then the fragment matching the length of
the target gene was recovered. Finally, validation by Illumina HiSeqTM
2,500 sequencing indicated that the cricRNA library has been
successfully constructed.
2.4. circRNA quantification and differential expression
After removing reads, including adapters, via screening raw reads
obtained by high-throughput sequencing, low-quality reads with >50%
useless bases and reads with more than 10% of unidentified nucleotides
were removed. The remaining unfiltered reads were prepared for circRNA
identification; 20mers from both ends of the unmapped reads were
extracted and aligned with the reference genome to detect unique anchor
positions within the splice site. Anchor reads aligning in the reversed
orientation (head-to tail) indicated circRNA splicing and were then
subjected to find_circ to identify circRNAs. Reads identified as
circRNAs are further expanded so that the circRNA sequence inside the
breakpoint of the read could be complete and surrounded by GU/AG splice
sites on both sides of the breakpoint. For quantifying circRNAs,
back-spliced junction reads were scaled to RPM (reads per million
mapped reads), and the formula was shown as follows:
[MATH:
RPM=10<
mn>6CN :MATH]
Where C is the number of back-spliced junction reads that are uniquely
aligned to a circRNA and N is the total number of back-spliced junction
reads.
Therefore, the calculated expression can be directly used for comparing
the differential expression among samples.
2.5. Gene ontology and Kyoto encyclopedia of genes and genomes pathway
enrichment analysis
The GO and KEGG pathway enrichment analyses followed procedures
described previously ([75]32). GO enrichment analysis provided all GO
terms for which source genes are significantly enriched compared to the
genomic background and filtered source genes corresponding to
biological functions. First, all source genes were mapped to GO terms
in the Gene Ontology database; gene numbers were calculated for every
term, and the significant GO enrichment for GO items compared to the
reference genome was analyzed using the hypergeometric test. The
formula for calculating the p-value is as follows:
[MATH: P=1−∑i=0
M−1MiN−Mn
−iNn
:MATH]
Here N is the number of all genes with GO/KEGG annotation; n is the
number of source genes in N; M is the number of all genes that are
annotated to the certain GO/KEGG terms.
KEGG pathway enrichment analysis was performed with reference to the GO
enrichment analysis method. Pathway enrichment analysis identified
significantly enriched metabolic pathways or signal transduction
pathways in source genes compared with the entire genome background.
The calculation formula is the same as that in the GO analysis.
2.6. miRNA sponge analysis and integrated analysis of circRNAs-miRNAs-mRNAs
For circRNAs (samples collected at 9 h) annotated in circBase, the
target relationship with miRNAs can be predicted using StarBase
(version 2.0). Mireap, Miranda (version 3.3a), and TargetScan (version
7.0) were used to predict sample target genes for novel circRNAs. To
predict mRNAs interacting with circRNAs and miRNAs, miRTarBase (v6.1)
was used to predict mRNAs targeted by the miRNAs sponge. The resulting
correlation of circRNAs-miRNAs-mRNAs can be visualized by Cytoscape.
2.7. Western blot and RT-qPCR
DEF cells were infected at 1 MOI to examine the expression of NDV
nucleocapsid protein (NP). Cells harvested at the indicated time points
were washed three times with PBS and lysed with RIPA buffer. Proteins
were separated under denaturing conditions in 10% sodium dodecyl
sulfate (SDS, Beyotime, China) polyacrylamide gels using a minigel
system, then transferred to nitrocellulose (NC) membranes. The membrane
was blocked for 1 h with 5% skimmed milk powder before being incubated
with anti-NP antibodies (NP antibody was preserved in our laboratory)
overnight at 4°C. After washing three times with PBS-Tween and once
with PBS, blots were incubated with peroxidase (POD) labeled
species-specific anti-immune globulin G (IgG) conjugates for 1 h at
room temperature. After washing four times as mentioned above, the NC
membrane was scanned and photographed by the Odyssey infrared imaging
system.
2.8. Nucleoplasm isolation and RNA extraction
Samples were subjected to nucleocytoplasmic separation according to the
instructions. RNA was extracted from cells using Trizol reagent.
2.9. Detection of circular RNA expression via relative quantitative RT-qPCR
RNA (1,000 ng) was used as a template, and Reverse Transcriptase M-MLV
reverse transcriptase (TaKaRa, Dalian, China), Random Primers(TaKaRa,
Dalian, China) and other reagents were added. Samples were kept in a
42°C constant-temperature water bath for 10 min and inactivated for
2 min at 95°C, cDNA was obtained and stored at −20°C. Primers were
designed according to the serial number of the GenBank duck source gene
([76]Table 1). Divergent primers were designed based on the circRNA
predicted sequences provided by the transcriptome. Amplification was
performed with ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing,
China) by RT-PCR. After the reaction, transcription level was
calculated by formula 2-(ΔΔct), and then the values were drawn into a
histogram by GraphPad Prism 8 software (GraphPad Software, Inc., United
States), and a difference analysis was performed.
Table 1.
Primers used in this study.
Primer Sequence (5′ → 3′) Length of product
ACTB-F CCCCATTGAACACGGTATTGTC 199 bp
ACTB-R GGCTACATACATGGCTGGGG
con-000094-F GGGAGGAATGGGGAGATAGG 299 bp
con-000094-R GTGATGACTGGTGAACGGGC
div-000094-F GGACTTGCCCGTTCACC 158 bp
div-000094-R TTCGTCGCCTCTTGCTG
con-000912-F TGTTGCTTTGCTTGTGACTCTG 118 bp
con-000912-R TTAAGTCCATGCGGGTTCTGA
div-000912-F CACAGTTTCTTGCTGACACAGAGA 119 bp
div-000912-R GCAAACAGCAAGGCTTCCCA
con-001672-F AAGATGCCCAACGTCTTCCA 113 bp
con-001672-R TGGAGTATGTCGAGGGCTGA
div-001672-F GCCTCCACACATTGACTATT 100 bp
div-001672-R CAACTCTCTTTCCATCTCGT
con-000030-F TCAAGTCGTCCTAGCCCAGT 345 bp
con-000030-R AGAGCAGCACAGCTACAAGG
div-000030-F CTTCAGGAAGAGATGGCCTGG 155 bp
div-000030-R TGTTGTGTCACCCACGTAGC
con-000991-F TCCTTCGTTTACAGCCGGTC 337 bp
con-000991-R AGAGCACACCTCTCTGGAGT
div-000991-F TAAGACCACCCGCGCTAGAA 147 bp
div-000991-R GTTAGCTGGCTTGTTGGTGT
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2.10. Sanger sequencing (circRNA)
The reverse-spliced region of circRNA was amplified using divergent
primers. The products recovered were sent to Sangon Biotech for Sanger
sequencing, and the sequencing results were aligned by NCBI Blast
analysis, MegAlign, and SnapGene.
2.11. siRNA interference test
For transfection, cells cultured in 6-well plates were washed 3 times
with PBS and transfected with siRNA ([78]Table 2) using Lipofectamine
2000 (Invitrogen) according to the manufacturer’s protocol.
Table 2.
SiRNAs for transfection.
siRNA Sequence (5′ → 3′)
si-circFBXW7-1 sense CACAAAGACAAAGAAUGUGTT
si-circFBXW7-1 antisense CACAUUCUUUGUCUUUGUGTT
si-circFBXW7-2 sense CAAAGACAAAGAAUGUGAATT
si-circFBXW7-2 antisense UUCACAUUCUUUGUCUUUGTT
si-circFBXW7-3 sense GACAAAGAAUGUGAAAGCATT
si-circFBXW7-3 antisense UGCUUUCACAUUCUUUGUCTT
NC sense UUCUCCGAACGUGUCACGUTT
NC antisense ACGUGACACGUUCGGAGAATT
[79]Open in a new tab
2.12. Statistical analysis
Data presentation and statistical analyses were performed using
GraphPad Prism (version 5.0; GraphPad Software, Inc., La Jolla, CA,
United States). Data were expressed as mean ± standard deviation (SD).
Data were analyzed using Student t-test for pairwise comparisons or an
analysis of variance/Dunn multiple comparison test for multiple
comparisons. Statistical significance was set at p < 0.05, p < 0.01,
and p < 0.001 for values that were significant, very significant, and
highly significant, respectively.
3. Results
3.1. Characterization of the circRNA expression profile post-NDV infection in
DEFs
The expression of NDV NP protein was detected by Western Blot and
indirect immunofluorescence assay ([80]Supplementary Figure S1),
indicating that NDV was able to infect duck embryo fibroblast (DEF)
cells. The indirect immunofluorescence assay was performed according to
the method of Sun et al. ([81]33). Lesions appeared after infecting DEF
cells with NDVs for 9 h with 1 multiplicity of infection (MOI)
([82]Figure 1); thus, 9 h post-NDV infection was selected as the
transcriptome sequencing time point. Three replicate cell samples were
selected from the control and experimental groups for sequencing. The
control groups (DEF cells without infection of NDV) were DN-1, DN-2,
and DN-3, while the experimental groups (DEF cells 9 h post-NDV
infection) were DK-1, DK-2, and DK-3. The samples were subjected to
transcriptome sequencing and purified to filter out the data, and 2,517
circRNAs were identified in the DEF cells. The number of circRNA
intersections between the control and infection group was 1,546
([83]Figure 2A). Candidate circular RNAs were similar to the control
group in length, chromosomal distribution, and type following NDV
infection in DEF cells. These candidate circRNAs were mainly located in
the exon region; the fewest circRNAs were mapped to the intergenic exon
regions in DEF cells, respectively ([84]Figure 2C). circRNAs of length
401–500 bp and 1,801–1,900 bp were the highest and the lowest,
respectively, in DEF cells, and no circRNAs had a length of < 100 bp
and no more than 2,000 bp ([85]Figure 2D). The results of circRNA
chromosomal distribution in DEF cells showed that most circRNAs were
distributed on chromosome 1, followed by chromosomes 2 and 3; no
circRNA was distributed on chromosome 17; > 50% of circRNAs were
distributed on chromosome 1–5 ([86]Figure 2B). Overall, circRNAs were
identified in DEF cells, and NDV infection could affect their
expression in DEF cells.
Figure 1.
[87]Figure 1
[88]Open in a new tab
Duck embryo fibroblast (DEF) cell lesions infected with Newcastle
disease virus (NDV) (100 μm). Control: DEF cells were seeded for 9 h
and micrographed; rGM: DEF cells were inoculated with the NDV rGM
strain for 9 h and micrographed.
Figure 2.
[89]Figure 2
[90]Open in a new tab
circRNA expression profile in duck embryo fibroblast (DEF) cells. (A)
Expressed circRNAs in DEF cells with or without NDV infection. (B)
Expressed circRNAs on each chromosome in DEF cells. (C) The different
circRNAs expressed in DEF cells. (D) The length of expressed circRNAs
in DEF cells.
3.2. Identification and verification of significantly DE circRNAs
To investigate the antiviral response to NDVs in DEF cells, the circRNA
expression profile was analyzed and significantly differentially
expressed circRNAs (fold change >2 and p-value <0.05) were screened; a
total of 57 (23 upregulated and 34 downregulated) genes were identified
([91]Supplementary Table S1). Significantly differentially expressed
circRNAs were identified in the DN group (non-NDV-infected DEF cells)
vs. the DK group (NDV-infected DEF cells). These results demonstrate
that circRNAs expressed in DEF cells show significant variations
post-NDV infection.
In the NDV-infected DEF cell group, five significantly differentially
expressed circRNAs, including three upregulated (novel_circ_000094,
novel_circ_001672, and novel_circ_000912) and two downregulated
(novel_circ_000991 and novel_circ_000030) circRNAs, were validated
([92]Table 3) using reverse transcription PCR analysis, Sanger
sequencing, and RNase R digestion tests. We designed converging and
diverging primers to validate circRNAs; the specific sites differed and
so did the size of the PCR products. Reverse transcription PCR analysis
demonstrated that the fragment of interest could not be amplified using
divergent primers with gDNA as a template ([93]Figure 3A, lane 4),
whereas the other lanes contained the amplified band of interest
([94]Figure 3A, lanes 1–3), suggesting that circRNAs were circular
structures derived from post transcriptional mRNA precursors via
alternative splicing. Sanger sequencing of the products after
amplification with divergent primers using cDNA as the template
revealed that the sequencing results were consistent with the back
splicing region alignment ([95]Figure 3B), further indicating that the
5 circRNAs selected for validation were reliable. Furthermore, qRT-PCR
validation using RNase R treated RNA showed that the CQ value of
β-actin was significantly higher than that of the untreated group,
indicating that the amount of housekeeping gene mRNA was significantly
reduced following RNase R treatment, whereas the circRNAs did not
change significantly after RNase R digestion ([96]Figure 3C),
indicating that the five selected circRNAs were all RNase R enzyme
tolerant with stable circular structures. The qRT-PCR results indicated
that the three upregulated and two downregulated circRNAs were
consistent with the transcriptome sequencing results in NDV-infected
DEF cells ([97]Figure 3D). These results confirmed circRNA
identification in this study as accurate and reliable.
Table 3.
circRNAs selected for expression verification.
circRNA Host source gene Length Type p-value 丨log2FC丨
Up/down-regulation
novel_circ_000912 CUL1 (ncbi_101793264) 937 bp annot_exons 0.04 18.57
Up
novel_circ_001672 NLK (ncbi_101804891) 221 bp exon_intron 0.04 18.5 Up
novel_circ_000094 FBXW7 (ncbi_101799234) 558 bp one_exon 0.03 2.26 Up
novel_circ_000991 BRF1 (ncbi_101796246) 539 bp annot_exons 0.03 1.88
Down
novel_circ_000030 ETNK1 (ncbi_101801722) 544 bp annot_exons 0.03 1.26
Down
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Figure 3.
[99]Figure 3
[100]Open in a new tab
Verification of five significantly differentially expressed circRNAs in
the NDV-infected DEF cell group. (A) Electrophoretogram of the PCR
products. M: DNA marker 500; 1: Convergent primers were used for
amplification using cDNA as template; 2: Divergent primers were used
for amplification using cDNA as template; 3: Convergent primers were
used for amplification using gDNA as template; 4: Divergent primers
were used for amplification using gDNA as template. (B) The
head-to-tail junctions of significantly differentially expressed
circRNAs were confirmed via Sanger sequencing. The red line indicates
back-splicing site position. The area from left (or right) of the site
is the end (or start) sequence of the circRNAs (junction). (C) qRT-PCR
was used to detect significantly differentially expressed circRNA
resistance to RNase R digestion. β-actin was used as a linearity
control. Data are expressed as means ± SEM (n = 3). (D) Validation of
significantly differentially expressed circRNAs in the NDV-infected DEF
cell group using qRT-PCR. Data are expressed as means ± SEM (n = 3).
3.3. KO and KEGG enrichment analyses of significantly DE circRNA
To further explore the functions of significantly differentially
expressed circRNAs produced post-NDV infection in DEF cells, the host
source genes of these circRNAs were used for GO and KEGG enrichment
analyses. For GO-molecular function (MF), GO-cellular component (CC),
and GO-biological process (BP), the significantly differentially
expressed circRNAs were mainly associated with binding and catalytic
activity, cell, cell part, and organelle, and cellular,
single-organism, and biological processes, respectively, post-NDV
infection in DEF cells ([101]Figure 4). GO enrichment analysis
indicated that the circRNAs in DEF cells might respond to NDV infection
by regulating different gene functions.
Figure 4.
[102]Figure 4
[103]Open in a new tab
Gene ontology (GO) annotation of significantly differentially expressed
circRNA from host source genes and Newcastle disease virus
(NDV)-infected duck embryo fibroblast (DEF) cells.
KEGG enrichment analysis showed that the significantly differentially
expressed circRNAs in NDV-infected DEF cells were mainly enriched in
the FoxO, MAPK, and mTOR signaling pathways, ubiquitin-mediated
proteolysis, and adherens junction ([104]Figure 5A). Additionally,
circRNAs were mainly enriched in environmental information
processing-related pathways ([105]Figure 5B). After NDV infection of
DEF cells, circRNA source genes were mainly involved in regulating
relevant central pathways in apoptosis-related pathways (FoxO and mTOR
signaling), cell growth pathways (FoxO signaling), and protein
degradation-related pathways (ubiquitin-mediated proteolysis),
suggesting that circRNAs in DEF cells might respond to NDV infection by
affecting signal pathways to relevant immune responses. Conclusively,
after NDV infection, DEF cells may mediate the molecular mechanism to
activate antiviral responses. In the NDV-infected DEF cell group, the
host source genes of circRNA novel_circ_001672 and novel_circ_000094,
extensively involved in multiple regulatory processes, were the most
important.
Figure 5.
[106]Figure 5
[107]Open in a new tab
Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation of
significantly differentially expressed circRNA. (A) Gradient map for
KEGG pathway annotation of significantly differentially expressed
circRNAs from Newcastle disease virus (NDV)-infected duck embryo
fibroblast (DEF) cells. (B) Circular map for KEGG pathway annotation of
significantly differentially expressed circRNAs from NDV-infected DEF
cells.
3.4. Establishment of circRNA-miRNA-mRNA interaction networks
circRNAs function as miRNA sponges and inhibit target gene degradation
by miRNAs mediating different biological processes during virus-induced
cancer and infection ([108]25, [109]34). In total, 106 bound miRNAs
were predicted in significantly differentially expressed circRNAs of
DEF cells with NDV infection. The miRNA genes binding mRNAs were
predicted to construct circRNA-miRNA-mRNA interaction networks; DEF
cells targeted multiple genes related to the central pathway or
apoptosis, including CASP8, BAK1, PIK3CA, MAPL3K14, and TRAF1
([110]Figure 6). These results suggest that DEF cells combat NDV
infection by regulating cellular pathways or apoptosis through
circRNA-targeted mRNAs and miRNAs. Among these, the above-mentioned
mRNAs, such as CASP8, BAK1 and PIK3CA, were mainly targeted by
mir-338-y, while novel_circ_000094 targeted mir-338-y, indicating a
novel_ circ_ 000094 that might have potential to regulate relevant
antiviral immune processes.
Figure 6.
[111]Figure 6
[112]Open in a new tab
circRNA-miRNA-mRNA interaction network of significantly differentially
expressed circRNAs from NDV-infected DEF cells.
3.5. circRNA-FBXW7 affected infection in NDV-infected DEF cells
The previous enrichment and interaction network results revealed that
novel_circ_000094 (circ-FBXW7) regulated multiple pathways and
functional genes during NDV infection. The validation results for these
genes were consistent with the sequencing results. Therefore, this
circRNA was selected to determine whether DEF cells resisted NDV
infection through it. Plasmids and siRNAs targeting circ-FBXW7 were
constructed and designed, respectively ([113]Figures 7A,[114]B). And we
found that si-3 for the circ-FBXW7 knockdown effect is the highest, and
the following experiments were carried out using this siRNA. The effect
of NDV proliferation after circ-FBXW7 knockdown or overexpression was
investigated; western blot and qRT-PCR results indicated that
circ-FBXW7 overexpression and knockdown suppressed ([115]Figures
7C,[116]E) and promoted ([117]Figures 7D,[118]F) NDV NP (Nuclear
protein) protein expression and gene transcription levels. NDV viral
growth curves showed similar results, with significantly lower and
higher viral titers at 12 h post-infection after circ-FBXW7
overexpression and knockdown, respectively ([119]Figures 7G,[120]H).
These results were consistent with the transcriptome sequencing
results, suggesting that DEF cells combat NDV infection by circ-FBXW7.
Figure 7.
[121]Figure 7
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Newcastle disease virus (NDV) proliferation after circ-FBXW7
overexpression or knockdown. (A) qRT-PCR verification of circ-FBXW7
overexpressed plasmid expression. (B) qRT-PCR verification of the siRNA
knockdown efficiency for circ-FBXW7. (C) NP gene production post-NDV
infection with circ-FBXW7 overexpression determined via qRT-PCR. (D) NP
gene expression post-NDV infection with circ-FBXW7 knockdown. (E) NP
protein production post-NDV infection with circ-FBXW7 overexpression.
(F) NP protein production post-NDV infection with circ-FBXW7 knockdown.
(G) Virus growth kinetics of NDV with circ-FBXW7 overexpression. The
NDV rGM strain was inoculated after transfection with the pCD-FBXW7
plasmid for 24 h in DEF cells. (H) Virus growth kinetics of NDV with
circ-FBXW7 knockdown.
3.6. KEGG enrichment analyses of circ-FBXW7-targeted mRNAs
Our study had demonstrated that circ-FBXW7 promoted resistance to NDV
infection in DEF cells. Enrichment analysis of mRNAs targeted by miRNAs
predicted to bind circ-FBXW7 was performed. Circ-FBXW7 was predicted to
bind mir-338-y, targeting 140 mRNAs. These 140 target genes were
subjected to KEGG enrichment analysis, which showed that targeted mRNAs
were mainly enriched in the cell cycle and senescence signaling
pathways ([123]Figure 8), indicating that DEF cells might combat NDV
infection by forming circ-FBXW7 combined with miRNAs targeting mRNAs to
regulate different process.
Figure 8.
[124]Figure 8
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KEGG enrichment analysis of mRNA targeted by miRNA binding with
circ-FBXW7.
4. Discussion
Genotype VII NDV strains are widespread in China ([126]2). The NDV host
range is wide and includes over 250 avian domestic and wild species
([127]34). Waterfowls are natural NDV hosts or carriers but resist NDV
infection ([128]35). Continuous outbreaks of genotype VII NDV-induced
ND in waterfowl have occurred since 1997 ([129]36). Elbestawy et al.
reported that the morbidity, mortality and viral shedding rates of
chickens and ducks infected with VII NDV differ ([130]9). Barber et al.
reported a variation between chicken and duck innate immune responses,
which exerted a more efficient interferon (IFN) response against viral
infection via RIG-I (absent in chickens) ([131]37), possibly
contributing to the differences in susceptibility to NDV and the
different clinical manifestations post-NDV infection between chickens
and ducks. Several studies have shown that chickens and waterfowls
combat NDV infection differently.
As covalently closed noncoding RNA, circRNAs are characterized by an
abundant expression, stable structure, and evolutionary conservation
([132]12). circRNA has been identified in several species
([133]14–[134]18); therefore, their expression profiles in birds,
including chickens and ducks (DEF cells), are similar. Several studies
have reported circRNA expression profiles in chicken, and the results
were similar to that of DEF cells in this study. In a duck study by Wu
et al., circRNAs in duck granulosa cells were mainly located in exons,
whereas circRNAs from duck preadipocytes in a study by Wang et al. were
mainly sense overlapping ([135]38, [136]39), indicating that the
circRNA expression profiles in ducks and other birds were relatively
conservative, and their length and circRNA source chromosomes were
similar to those of other birds; however, the circRNAs formed differed.
The circRNAs changes in this study might be due to the mechanism by
which DEF cells respond to NDV infection by forming different circRNAs
to resist NDV invasion.
GO and KEGG pathway enrichment analyses of circRNA source genes are
essential for exploring and predicting circRNA roles in NDV infection.
Several GO annotation analyses of significantly differentially
expressed circRNAs produced post-infection with different viruses
revealed that the host participated in the physiological activities of
various cells through circRNAs production ([137]40–[138]42). Consistent
with our results, GO annotation analysis revealed that significantly
differentially expressed circRNAs produced post-NDV infection in DEF
cells are abundantly expressed in various molecular-, biological-, and
cellular-related functions, indicating that circRNAs in DEF cells might
broadly involve in various physiological activities of cells in
response to NDV infection. Guo et al. found that chickens regulate the
innate immune response to produce antibodies via circRNA with LaSota
infection ([139]43). These results indicate that infection with NDV
could also affect innate immune response in vivo. However, Guo et al.
used lentogenic strain in their study, while we used virulent strain.
There might be some differences in the regulation of innate immune
response between virulent and lentogenic strain. Coupled with our KEGG
enrichment results, we speculated that DEF cells might affect
cell-related signaling pathways for antiviral responses to NDV
infection by regulating circRNA-induced ubiquitination progress. Hosts
target the HCV NS5A protein via ISG12a to mediate NS5A degradation via
the ubiquitination-dependent proteasome pathway for antiviral responses
([140]44). Consistent with our KEGG enrichment results, DEF cells may
utilize circRNAs to target the NDV protein degradation directly or
indirectly via the ubiquitination-dependent proteasome degradation
pathway, exerting antiviral responses against NDV. Guo et al. found
that the Japanese encephalitis virus (JEV) induces apoptosis by
inhibiting the STAT3-FOXO-BCL-6/p21 pathway ([141]45). Our KEGG
enrichment results showed that circRNAs in DEF cells post-NDV infection
were enriched in apoptosis and apoptosis-related pathways, suggesting a
similar antiviral mechanism in DEF cells, possibly by inducing
apoptosis to resist NDVs infection. Further analysis revealed that
caspase-8 and STAT1 genes, targeted by multiple significantly
differentially expressed circRNAs, were widely involved in antiviral
responses, growth inhibition, apoptosis, and other related biological
processes ([142]46, [143]47). These genes were mir-338-y-targeted,
suggesting that the differentially expressed circRNAs might regulate
gene expression mainly by affecting mir-338-y for antiviral responses
against NDV infection. Mir-338-y was only targeted by circ-FBXW7
(novel_circ_000094), and circ-FBXW7 regulated multiple pathways in the
KEGG enrichment, indicating that circ-FBXW7 may play an important role
in antiviral responses to NDV infection in DEF cells.
Circ-FBXW7 overexpression inhibited NDV infection, which was promoted
with circ-FBXW7 knockdown. Interestingly, this promotion and inhibition
of virus replication can be seen at 12 h post-infection, since our
sequencing results are based on the analysis of NDV infected samples at
9 h. Here, the circ-FBXW7 begins to function, hence significant
differences were detected at 12 h post-infection. One round of NDV
infection and replication takes about 6 h, indicating that circ-FBXW7
may not affect the invasion and replication stages of NDV virus. The M
protein of NDV is thought to be dependent on ubiquitination to play the
role of viral nucleocytoplasmic shuttling and release ([144]48,
[145]49). The circ-FBXW7-derived gene is thought to be involved in the
regulation of ubiquitination ([146]50). Therefore, we speculate that
DEF cells may use circ-FBXW7 to regulate the ubiquitination
modification process and affect the stability of M protein to achieve
the effect of inhibiting NDV virus budding and release. Circ-FBXW7 has
been widely studied, and its diverse functions have been suggested as
cognate circRNA from circ-FBXW7 has distinct functions. Yang et al.
demonstrated that FBXW7-185aa, encoded by the spanning junction open
reading frame in circ-FBXW7 driven by an internal ribosome entry site,
suppressed glioma tumorigenesis ([147]51). Lu et al. showed that
circ-FBXW7 regulates cancer cell generation and metastasis by
inhibiting the NEK2 and mTOR signaling pathways and activating PTEN
([148]52). Ye et al. demonstrated that circ-FBXW7 acts as a mir-197-3p
sponge to inhibit the proliferation and migration of triple-negative
breast cancer cells by increasing linear FBXW7 mRNA expression and
inducing c-Myc degradation ([149]53). Moreover, in a rectal cancer
study, circ-FBXW7 was found in exosomes, which caused resistant cell
sensitization to oxaliplatin, increased oxaliplatin-induced apoptosis,
and inhibited oxaliplatin-induced epithelial-mesenchymal transition
([150]54). These findings suggest that circ-FBXW7 exhibits diverse
functions in different cancer processes; however, circ-FBXW7 exerting
antiviral functions required elucidation. Therefore, to understand the
interaction network between circ-FBXW7 and mir-338-y, KEGG enrichment
analysis of mir-338-y-regulated target genes, mainly involved in cell
cycle-related pathways, was performed. Cell cycle regulation plays an
important role in cell growth and viral infection progression; thus,
circ-FBXW7 may exert antiviral effects by targeting mir-338y to
regulate cell cycle-related genes.
The circRNA produced by the virus during infection have also been
extensively studied. Our study revealed that NDV infection of DEF cells
altered circRNA expression profiles, while the significantly
differentially expressed circRNA exerted different functions via
multiple cellular processes and related pathways. Additionally, we
investigated the production process of hosts to produce circRNA to
combat viral infection. Circ-FBXW7 might have played a role in
antiviral responses in DEF cells infected with NDV. However, the
mechanism and related pathways remain to be determined. Finally, the
formation and interaction mechanisms between viral circRNA and host
circRNAs remain to be fully investigated. In addition, it is potential
to study the regulation of innate immunity by NDV infection in vivo of
waterfowl.
Data availability statement
The datasets presented in this study can be found in online
repositories. The names of the repository/repositories and accession
number(s) can be found at: [151]https://www.ncbi.nlm.nih.gov/geo/,
[152]GSE229335.
Author contributions
LF participated in all the experiments and manuscript drafting. JR, YW,
and YYC collected data and created the pictures. YCC assisted with the
experiment. LC analyzed the experimental data. QL, ML, and CD reviewed
the article. BX and TR helped to design the study. All authors
contributed to the article and approved the submitted version.
Funding Statement
This research was funded by the National Natural Science Foundation of
China (no. 31902251), the Guangdong Provincial Special Fund for Modern
Agriculture Industry Technology Innovation Teams (no. 2022KJ119), the
National Natural Science Foundation of China (no. 31872492) and the
Special Project on Strategy for Rejuvenation of Guangdong Provincial
villages in 2022 (no. 5500-F22039).
Conflict of interest
The authors declare that the research was conducted in the absence of
any commercial or financial relationships that could be construed as a
potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or claim
that may be made by its manufacturer, is not guaranteed or endorsed by
the publisher.
Supplementary material
The Supplementary material for this article can be found online at:
[153]https://www.frontiersin.org/articles/10.3389/fvets.2023.1181916/fu
ll#supplementary-material
[154]Click here for additional data file.^ (10.3KB, xls)
[155]Click here for additional data file.^ (323.7KB, TIF)
References