Identification and analysis of long non-coding RNAs that are involved in inflammatory process in response to transmissible gastroenteritis virus infection
- 204 Downloads
Transmissible gastroenteritis virus (TGEV) infection can cause acute inflammation. Long noncoding RNAs (lncRNAs) play important roles in a number of biological process including inflammation response. However, whether lncRNAs participate in TGEV-induced inflammation in porcine intestinal epithelial cells (IPECs) is largely unknown.
In this study, the next-generation sequencing (NGS) technology was used to analyze the profiles of lncRNAs in Mock and TGEV-infected porcine intestinal epithelial cell-jejunum 2 (IPEC-J2) cell line. A total of 106 lncRNAs were differentially expressed. Many differentially expressed lncRNAs act as elements to competitively attach microRNAs (miRNAs) which target to messenger RNA (mRNAs) to mediate expression of genes that related to toll-like receptors (TLRs), NOD-like receptors (NLRs), tumor necrosis factor (TNF), and RIG-I-like receptors (RLRs) pathways. Functional analysis of the binding proteins and the up/down-stream genes of the differentially expressed lncRNAs revealed that lncRNAs were principally related to inflammatory response. Meanwhile, we found that the differentially expressed lncRNA TCONS_00058367 might lead to a reduction of phosphorylation of transcription factor p65 (p-p65) in TGEV-infected IPEC-J2 cells by negatively regulating its antisense gene promyelocytic leukemia (PML).
The data showed that differentially expressed lncRNAs might be involved in inflammatory response induced by TGEV through acting as miRNA sponges, regulating their up/down-stream genes, or directly binding proteins.
KeywordsTGEV lncRNAs miRNAs lncRNA binding proteins
competing endogenous RNA
coding potential calculator
DExD/H-Box helicase 58
dulbecco’s modified eagle medium (DMEM)/F-12/HAM
false discovery rate
fragments per kilobase of transcript per million mapped reads
inhibitor of DNA binding 2
interferon induced protein with tetratricopeptide repeats 5
interferon beta 1
porcine intestinal epithelial cells-jejunum 2
porcine intestinal epithelial cells
interferon regulatory factor 1
kyoto encyclopedia of genes and genomes
long intervening/intergenic non-coding RNAs
long non-coding RNAs
minimum free energy
multiplicity of infection
reference annotation-based transcripts
radical S-Adenosyl methionine domain containing 2
small cajal body-specific RNA 13
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
signal transducer and activator of transcription 1
T cell receptors
transmissible gastroenteritis virus
tumor necrosis factor
tripartite motif containing 25
Virus can activate the inflammatory response by multiple means, including Nuclear factor-kappa B (NF-κB), Jak-STAT, TLRs, T cell receptors (TCRs), NLRs, TNF, RLRs signaling pathway [1, 2, 3, 4, 5, 6, 7]. Previous studies have described that TGEV can impair IPECs and trigger inflammatory response . IPECs are the targets for TGEV, and play an important role in the nutrition absorption and inflammatory response against pathogens. The pathogenesis of TGEV is strongly associated with the powerful induction of inflammatory response in host cells. A new study confirmed that the RLRs, TLRs and NF-κB signaling pathways are involved in TGEV-induced inflammatory responses .
Non-coding RNAs (ncRNAs), including miRNAs, circular RNAs (circRNAs), as well as lncRNAs, typically do not encode proteins and functionally regulate many biological process . It has been demonstrated that many ncRNAs are involved in inflammatory response in cells [2, 3, 11, 12, 13, 14, 15]. In previous study, we determined that the profiles of mRNAs, miRNAs and circRNAs were significantly changed in the IPEC-J2 after TGEV infection. The potential functions of differentially expressed mRNAs, miRNAs and circRNAs were anlyzed and were closely related to inflammatory response . Recently, increasing studies have indicated that lncRNAs play important roles in inflammatory response [17, 18, 19, 20]. Therefore, we proposed that lncRNAs also might participate in regulating inflammatory response during TGEV infection.
The lncRNAs play roles in regulating transcription, translation, and protein translocation [21, 22, 23, 24, 25]. LncRNAs can regulate translation by interacting with miRNA or act as precursors of miRNA [26, 27, 28]. For example, lncRNA SBF2-AS1 acts as a competing endogenous RNA (ceRNA) to modulate cell proliferation via binding with miR-188-5p in acute myeloid leukemia . LncRNA HOTAIR functions as a ceRNA to upregulate Sirtuin 1 (SIRT1) by sponging miR-34a in diabetic cardiomyopathy . LncRNAs can serve as scaffold to bind to different types of proteins or transcription factors at specific domains to activate or inhibit gene transcription. LncRNA H19 decreases the transcriptional activity of p53 . LncRNA SNHG10 facilitates hepatocarcinogenesis and metastasis by modulating its homolog Small Cajal body-specific RNA 13 (SCARNA13) . LncRNAs can also achieve the regulation of the expression of the target genes by recruiting some RNA-binding proteins .
This is the first study to demonstrate the expression profiles and regulatory mechanisms of lncRNAs during TGEV infection by NGS methods. The data showed that differentially expressed lncRNAs might be involved in inflammatory response induced by TGEV through acting as miRNA sponges, regulating their up/down-stream genes, or directly binding proteins. This information will enable further research on the TGEV infection and facilitate the development of novel TGE therapeutics targeting lncRNAs.
Overview of the Solexa high-throughput sequencing data
Feature comparison of lncRNA and mRNA
Profiling of lncRNAs
LncRNAs don’t act as miRNA precursors when TGEV infected
LncRNAs act as miRNA sponges
Up- and down-stream genes of differentially expressed lncRNAs
Validation of lncRNAs by quantitative real time polymerase chain reaction (q RT-PCR)
Function analysis of the antisense lncRNA TCONS_00058367
LncRNAs have been reported to be involved in the coronavirus infections [20, 34], but the roles of lncRNAs during TGEV induced inflammation response have not yet been elucidated. In our study, NGS techniques were used to investigate the lncRNA expression profiles of TGEV infected IPEC-J2. Among the transcripts of IPEC-J2 obtained in our study, a total of 2023 lncRNAs across the entire genome were screened after sequencing and bioinformatics analysis. These lncRNAs were characterized by shorter transcript length, longer exons, lower estimated number of exons and lower expression levels. These properties were also observed in other reported lncRNAs within the genome [20, 35, 36, 37].
In a previous study, TGEV induced inflammatory response via NF-κB signaling pathway, TLRs signaling pathway, NLRs signaling pathway, Jak-STAT signaling pathway, TNF signaling pathway and RLRs signaling pathway . In our study, We identified 106 lncRNAs differential expression between TGEV-infected group and Mock-infected group, reminding us that lncRNAs may be involved in the regulatory process of TGEV infection. LncRNAs can rescue the translation levels of mRNA via pairing to miRNAs to prevent the binding of miRNAs and mRNA UTR. In this study, we found mir-218, which we mentioned earlier, had three target genes, DExD/H-Box helicase 58 (DDX58), Interferon Regulatory Factor 1 (IRF1) and Signal Transducer And Activator Of Transcription 1 (STAT1) that might be involved in inflammatory response. Additionally, ten lncRNAs TCONS_00002283, TCONS_00019226, TCONS_00019227, TCONS_00021915, TCONS_00037709, TCONS_00043977, TCONS_00052757, TCONS_00064461, TCONS_00067143 and TCONS_00067979, which were differentially expressed in TGEV-infected group, were predicted to be targeted by this miRNA, indicating that the lncRNAs may compete with DDX58, IRF1 and STAT1 to affect their expression levels and influence TGEV-induced inflammatory response. Some lncRNAs can directly bind to proteins to regulate the functions of proteins [25, 38]. We determined lncRNA-protein interactions using the catRAPID omics algorithm, the result showed that 34 lncRNAs interacted with 4 proteins, including C7, ID2, MYC, and IRF1, which involve in immune system process. One of the important functions of lncRNA is to act as antisense transcripts of mRNAs or located adjacent to protein coding genes. In our data, many neighbouring genes correspond to compartments of the inflammatory response, such as PML (ENSSSCT00000002141), Interferon Beta 1 (IFNB1) (ENSSSCT00000005691), Radical S-Adenosyl methionine domain containing 2 (RSAD2) (ENSSSCT00000009461), and interferon induced protein with tetratricopeptide repeats 5 (IFIT5) (ENSSSCT00000011440). Previous studies have shown that NF-κB signaling pathway, one of the most important pathways, plays an important role during TGEV- induced inflammatory response [9, 16, 39, 40]. Therefore, changes in the expression levels of genes, which related in NF-κB signaling pathway, might influence the TGEV-induced inflammatory response. The differentially expressed lncRNAs may affect TGEV-induced inflammatory response by affecting NF-κB signaling pathway. It has been proved that PML promotes TNF-α-induced transcriptional responses by promoting NF-κB activity . We further confirm that silencing PML gene expression rescued the TGEV-induced NF-κB activity. In our study, lncRNA TCONS_00058367 was identified as a potential antisense transcript of PML, which suppress transcription of PML. Our work uncovered that lncRNAs might act as regulatory elements of the host inflammatory response when TGEV-infected. While, further efforts should be paied to confirm the present findings.
The lncRNA expression profile of IPEC-J2 was compared between the IPEC-J2 infected with TGEV (n = 2) and Mock group (n = 2). To identify lncRNAs expressed in TGEV infected IPEC-J2, cDNA libraries were constructed and sequenced on the HiSeq 2500 Illumina platform (Illumina, San Diego, CA, USA).
Strand-specific library construction and sequencing
IPEC-J2 cells were infected with TGEV at 1 MOI for 24 h (indicated by T1 and T2). Meanwhile, the mock infection (indicated by M1 and M2) was carried out. Total RNA was extracted with Trizol reagent (Invitrogen, Carlsbad, CA, US). After total RNA was extracted, ribosomal RNAs (rRNAs) were removed to retain mRNAs and ncRNAs. Following the purification, the enriched mRNAs and ncRNAs were iron-fragmented at 95 °C. Then, reverse transcriptase and random primers were used to generate the first strand cDNA from the cleaved RNA fragments. The second strand DNA was amplified by PCR, QiaQuick PCR extraction kit was used to purify the cDNA fragments, then these fragments were end repaired, poly(A) added, and ligated to Illumina sequencing adapters. The second-strand cDNA was digested by uracil-N-glycosylase (UNG), the products were size selected by PCR amplified, agarose gel electrophoresis, and sequenced using Illumina HiSeqTM 2500 system (Illumina, USA).
Alignment with reference genome
Reads containing adapters, low quality reads, and rRNA reads were removed. The remaining reads of each sample were then mapped to Sus scrofa reference genome (Sus scrofa 10.2) by TopHat2 (version 126.96.36.199), respectively.
Cufflinks (V2.2.1), which preferring to the program reference annotation-based transcripts (RABT), was used to reconstruct the transcripts. The influence of low coverage sequencing was fixed through Cufflinks constructing faux reads based on reference. During the end of assembly, similar fragments were removed from all of the reassembled fragments by aligning with reference genes. Then we used Cuffmerge to merge transcripts from different replicates of a group into a comprehensive set of transcripts, and then the transcripts from multiple groups were merged into a finally comprehensive set of transcripts.
Identification and annotations for novel transcripts
To identify the novel transcripts, all of the reconstructed transcripts were aligned with reference genome and divided into twelve categories using Cuffcompare (V2.2.1). We used the following parameters to identify reliable novel transcripts: the length of transcript was longer than 200 bp and the exon number was more than 2.
Classification, characterization, and validation of lncRNAs
Two softwares coding-non-coding index (CNCI) (https://github.com/www-bioinfo-org/CNCI)  and coding potential calculator (CPC) (http://cpc.cbi.pku.edu.cn/)  were used to assess the protein-coding potential of new transcripts by default parameters. The intersection of both results were chosen as long non-coding RNAs.
Quantification of lncRNA abundance
C, the number of fragments that are mapped to transcripts; N, the total number of fragments that are mapped to reference genes; L, the number of base pairs of transcript.
Significance analysis of lncRNAs
The edgeR package (http://www.r-project.org/) was used to identify differentially expressed lncRNAs. A fold change ≥2 and ≤ 0.5, plus a false discovery rate (FDR) <0.05, were identified as significant differentially expressed lncRNAs.
miRNA precursor prediction
LncRNAs can be spliced into multiple small RNAs which function as post-transcriptional regulators. To find potential miRNA precursors, lncRNAs were aligned to miRBase (version 21). Those with identity more than 90% were selected.
Based on the sequences of lncRNAs, three softwares RNAhybrid (v2.1.2) + svm_light (v6.01), Miranda (v3.3a) and TargetScan (Version:7.0) were used to the candidate target genes. The interaction networks among lncRNA and miRNA were built and visualized using Cytoscape (v3.5.1) (http://www.cytoscape.org/).
LncRNA cis-regulation analysis
One of the functions of lncRNAs is cis-regulation of their neighboring genes on the same allele. The up-stream lncRNAs which have intersection of promoter or other cis-elements may regulate gene expression in transcriptional or post-transcriptional level. The downstream or 3’UTR region lncRNAs may have other regulatory functions. LncRNAs, which are classified as located in an “unknown region” in Cuffcompare (V2.2.1) were annotated as up-or downstream of a gene. LncRNAs in up/down stream of a gene were likely to be cis-regulators. The interaction networks among lncRNA and up-or downstream genes were built and visualized using Cytoscape (v3.5.1) (http://www.cytoscape.org/).
Antisense lncRNA analysis
In order to reveal the interaction between antisense lncRNA and mRNA, the software RNAplex  (http://www.tbi.univie.ac.at/RNA/RNAplex.1.html) was used to predict the complementary correlation of antisense lncRNA and mRNA.
GO and KEGG analysis of differentially expressed lncRNAs
GO database (http://www.geneontology.org/) and KEGG database (http://www.genome.jp/kegg/) were used to annotate the pathways. The calculating formula is the same as the previous study .The interaction networks among lncRNAs, miRNAs, mRNAs or proteins were built and visualized using Cytoscape (v3.5.1) (http://www.cytoscape.org/).
Quantification of lncRNAs, miRNAs, and mRNAs using qRT-PCR
According to the manufacturer′s instructions, TRIzol reagent was used to extract the total RNA of IPEC-J2 cells, then reverse transcription was carried out using M-MLV reverse transcriptase (Invitrogen, US). qRT-PCR was performed on iQ5 qRT-PCR System (Bio-Rad, US). The primers are shown in Additional file 10:Table S10.
Western blot analysis
RIPA lysis buffer containing phenylmethylsulfonyl fluoride (PMSF) was used to treat samples to extract the protein, then using BCA Protein Assay Reagent (Pierce, US) to measure the protein concentration. Proteins were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, US) subsequently. Block the PVDF membrane with 5% non-fat milk for 2 h at room temperature and then incubate the PVDF membrane with Phospho-NF-κB p65 (p-p65) Rabbit monoclonal antibody (CST, US) overnight at 4 °C and Horseradish peroxidase (HRP)-conjugated secondary antibody (Pierce, US) at room temperature for 1 h subsequently. In the last step, the membrane was developed with enhanced chemiluminescence (ECL) (Promega, US).
SPSS 16.0 was used for statistical analysis. The data are presented as the means ± SEM. Statistical significance was analyzed by unpaired Student′s t-test. p < 0.05 was defined as statistical significance.
We thank Zhanyong Wei of College of veterinary medicine, Henan Agricultural University, for gifting IPEC-J2 cell line.
XM performed the experiments, analyzed the data, wrote the paper, prepared figures and Tables. XZ conceived and designed the experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote the paper, prepared figures and tables, reviewed drafts of the paper. KW and XT were involved in drafting and revising the manuscript. JG and MM performed the experiments and analyzed the data. YQ and LC were involved in drafting the manuscript and prepared Figs. YH was involved in revising the manuscript. DT conceived and designed the experiments, contributed reagents/materials/analysis tools, reviewed drafts of the paper. All authors have read and approved the manuscript.
This work was supported by Key Research and Development Project in Shaanxi Province (Grant No. 2018ZDXM-NY-064), National Natural Science Foundation of China (Grant No. 31472167, 31972645), Science and Technology Planning Project of Yangling demonstration zone (Grant No. 2018NY-35), and Central Project of Major Agricultural Technology Promotion Funds (Grant No. K3360217060).
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
- 3.Yang L, Zhang YJ. Antagonizing cytokine-mediated JAK-STAT signaling by porcine reproductive and respiratory syndrome virus. Vet Microbiol. 2016.Google Scholar
- 16.Ma X, Zhao X, Zhang Z, Guo J, Guan L, Li J, Mi M, Huang Y, Tong D. Differentially expressed non-coding RNAs induced by transmissible gastroenteritis virus potentially regulate inflammation and NF-kappaB pathway in porcine intestinal epithelial cell line. BMC Genomics. 2018;19(1):747.PubMedPubMedCentralCrossRefGoogle Scholar
- 17.Lin H, Jiang M, Liu L, Yang Z, Ma Z, Liu S, Ma Y, Zhang L, Cao X. The long noncoding RNA Lnczc3h7a promotes a TRIM25-mediated RIG-I antiviral innate immune response. Nat Immunol. 2019.Google Scholar
- 21.D'Angelo D, Mussnich P, Sepe R, Raia M, Del Vecchio L, Cappabianca P, Pellecchia S, Petrosino S, Saggio S, Solari D, et al. RPSAP52 lncRNA is overexpressed in pituitary tumors and promotes cell proliferation by acting as miRNA sponge for HMGA proteins. Journal of molecular medicine (Berlin, Germany). 2019;97:1019–32.CrossRefGoogle Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.