Identification and analysis of long noncoding RNAs that are involved in inflammatory process in response to transmissible gastroenteritis virus infection

- Identification and analysis of long non- coding RNAs that are involved in.
- 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.
- 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)..
- Conclusions: 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..
- and inflammatory response against pathogens.
- Non-coding RNAs (ncRNAs), including miRNAs, cir- cular RNAs (circRNAs), as well as lncRNAs, typically do not encode proteins and functionally regulate many bio- logical process [10].
- It has been demonstrated that many ncRNAs are involved in inflammatory response in cells .
- 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0.
- The potential functions of differentially expressed mRNAs, miRNAs and circRNAs were anlyzed and were closely related to inflammatory response [16].
- LncRNAs can also achieve the regu- lation of the expression of the target genes by recruiting some RNA-binding proteins [32]..
- 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.
- Overview of the Solexa high-throughput sequencing data To investigate the lncRNA expression profiles of TGEV infected IPEC-J2, IPEC-J2 were infected with TGEV strain (TGEV-infected group, indicated by T1 and T2) and the normal IPEC-J2 line (Mock-infected group, indicated by M1 and M2) was used as a control.
- 1a and Additional file 1: Table S1).
- 1b and Add- itional file 2: Table S2).
- The average mRNA expression level was higher than that of the known lncRNAs and novel lncRNAs (Fig.
- Profiling of lncRNAs.
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- A total of 61 differentially expressed lncRNAs and 55 differentially expressed mRNAs tar- geted 11 differentially expressed miRNAs in the network respectively (Fig.
- 5a and Additional file 5: Table S5).
- To find the potential function of these significantly differen- tially expressed lncRNAs acting as miRNA sponges, kyoto encyclopedia of genes and genomes (KEGG) ana- lysis of the 55 differentially expressed mRNAs was per- formed and presented.
- Three hundred seventy- two lncRNA-protein interactions were predicted for differentially expressed lncRNAs (Fig.
- 6a and Add- itional file 6: Table S6).
- Up- and down-stream genes of differentially expressed lncRNAs.
- We predicted the up- and down-stream genes of dif- ferentially expressed lncRNAs (100 K).
- 1 Classification of the assembled transcripts of IPEC-J2 according to their Ensembl code class (pie graphs) detailing lncRNA distribution (bar graphs) of: (a) and (b) all expressed transcripts.
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- 7a and Additional file 7: Table S7.
- GO analysis was conducted to enrich up- and down- stream targets of differentially expressed lncRNAs (http://www.geneontology.org.
- The results exhibited that the 34 up- and down-stream targets of differen- tially expressed lncRNAs were primarily enriched in immune system process (Fig.
- To validate the RNA-seq results of differentially expressed lncRNAs, we tested the expression levels of them using qRT-PCR.
- 8 and Additional file 3: Table S3..
- Function analysis of the antisense lncRNA TCONS_00058367.
- The result showed that lncRNA TCONS_00058367 was located in physical contiguity PML (MFE.
- NF-κB signaling pathway plays an import- ant role during TGEV- induced inflammatory response..
- The antisense lncRNA TCONS_00058367 was down- regulated in TGEV-infected group, and PML was up- regulated in TGEV-infected group.
- 2 Genomic features of lncRNAs.
- a, b, d are standard boxplots, which display the distribution of data by presenting the inner fence (the whisker, taken to 1.5× the Inter Quartile range, or IQR, from the quartile), first quartile, median, third quartile and outliers.
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- The STRING database (version 10.0) was used to further understand the regulatory relationship between PML and other differentially expressed mRNAs related to inflamma- tion process (Fig.
- 9c and Additional file 8: Table S8).
- In our study, NGS techniques were used to investigate the lncRNA expression profiles of TGEV infected IPEC-J2.
- 3 Clustering and Heatmap analysis of differentially expressed lncRNAs (FPKM) across TGEV infection (T1, T2) and Mock infection (M1, M2)..
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- 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.
- 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 re- sponse, such as PML (ENSSSCT Interferon Beta 1 (IFNB1) (ENSSSCT Radical S- Adenosyl methionine domain containing 2 (RSAD2) (ENSSSCT and interferon induced protein with tetratricopeptide repeats 5 (IFIT5) (ENSSSCT .
- Previous studies have shown that NF-κB signaling pathway, one of the most import- ant pathways, plays an important role during TGEV- induced inflammatory response .
- There- fore, changes in the expression levels of genes, which related in NF- κ B signaling pathway, might influence the TGEV-induced inflammatory response.
- The differ- entially expressed lncRNAs may affect TGEV-induced.
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- inflammatory response by affecting NF-κB signaling pathway.
- Our work uncovered that lncRNAs might act as regulatory ele- ments of the host inflammatory response when.
- TGEV-infected.
- 5 Regulatory network analysis of lncRNA-miRNA-mRNA.
- b KEGG enrichment analysis of lncRNA-miRNA-mRNA.
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- 6 Regulatory network analysis of lncRNA-proteins.
- b GO enrichment analysis of the interacting proteins of lncRNAs.
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- During the end of assembly, similar fragments were removed from all of the reassembled fragments by aligning with reference genes.
- 7 Regulatory network analysis of up- and down-stream genes of lncRNAs.
- a The interaction network of lncRNAs and their up- and down- stream genes.
- b GO enrichment analysis of up- and down-stream genes of lncRNAs.
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- 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).
- Significance analysis of lncRNAs.
- The edgeR package (http://www.r-project.org/) was used to identify differentially expressed lncRNAs.
- ≥2 and ≤ 0.5, plus a false discovery rate (FDR) <0.05, were identified as significant differentially expressed lncRNAs..
- One of the functions of lncRNAs is cis-regulation of their neighboring genes on the same allele.
- 8 qRT-PCR validation of lncRNAs.
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- 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.
- The calculating formula is the same as the previous study [16].The interaction networks among lncRNAs, miRNAs, mRNAs or proteins were built and visu- alized using Cytoscape (v3.5.1) (http://www.cytoscape.org/)..
- 9 Function analysis of the antisense lncRNA TCONS_00058367.
- TCONS_00058367 were measured by qRT-PCR (normalized to β -actin and in reference to the control).
- c The regulatory relationship between PML and other differentially expressed mRNAs related to immune system process (red).
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- Additional file 1: Table S1.
- Classification of the assembled transcripts of IPEC-J2..
- Additional file 2: Table S2.
- Classification of the lncRNAs of IPEC-J2..
- Additional file 3: Table S3.
- The detailed information of differentially expressed lncRNAs..
- Additional file 4: Function analysis of the antisense lncRNA TCONS_00058367.
- Additional file 5: Table S5.
- Additional file 6: Table S6.
- Additional file 7: Table S7.
- Additional file 8: Table S8.
- Additional file 9: Table S9.
- 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.
- DT conceived and designed the experiments, contributed reagents/materials/analysis tools, reviewed drafts of the paper.
- The accession numbers of the TGEV-infected group (T1, T2) and the Mock- infected group (M1, M2) are No.SRR6447591 and No.SRR6447590..
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- Beyond the inflammasome: regulatory NOD-like receptor modulation of the host immune response following virus exposure.
- Control of the rescue and replication of Semliki Forest virus recombinants by the insertion of miRNA target sequences.
- 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.
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