- Unveiling the long non-coding RNA profile of porcine reproductive and respiratory syndrome virus-infected porcine alveolar macrophages. - By far the majority of studies have focused on the immune-related lncRNAs of mice and humans, but the function of lncRNAs in porcine immune cells are poorly understood. - Porcine reproductive and respiratory syndrome virus (PRRSV) impairs local immune responses in the lungs of nursery and growing pigs, whereas the virus triggers the inflammatory responses. - Results: In this study, a total of 350 annotated lncRNAs and 1792 novel lncRNAs in PAMs were identified through RNA-seq analysis. - Among them 86 differentially expressed (DE) lncRNAs and 406 DE protein-coding mRNAs were identified upon PRRSV incubation. - GO category and KEGG pathway enrichment analyses revealed that these DE lncRNAs and mRNAs were mainly involved in inflammation- and pathogen infection-induced pathways. - The results of dynamic correlated expression networks between lncRNAs and their predicted target genes uncovered that numerous lncRNAs, such as XLOC-022175, XLOC-019295, and XLOC-017089, were correlated with innate immune genes. - This study suggests that porcine lncRNAs affect immune responses against PRRSV infection through regulating their target genes in PAMs.. - In response to PRRSV infection, comprehensive DE lncRNAs and mRNAs were profiled from PAMs. - Keywords: Porcine reproductive and respiratory syndrome virus, Long non-coding RNA, Porcine alveolar macrophage, mRNA-lncRNA correlation network. - The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. - However, little is known regarding the function of lncRNAs in porcine innate immune cells during virus infection.. - Porcine reproductive and respiratory syndrome virus (PRRSV) is the etiologic agent of porcine reproductive and respiratory syndrome, which is characterized by re- spiratory problem in growing pigs and reproductive fail- ure in sows [15, 16]. - However, the intracellular regulatory mechanisms of lncRNAs related to these innate immune properties remain to be ad- dressed. - Expression profile of lncRNA in PAMs. - More specifically, our pool contained 2142 lncRNAs (350 known lncRNAs and 1792 novel lncRNAs) and 22,565 mRNAs. - The result showed that enriched GO and KEGG pathways were mainly related to the inflammatory response, such as. - “MAPK signaling pathway”, “cytokine-cytokine receptor interaction”, “TNF signaling pathway”, “toll-like receptor signaling pathway”, and “Jak-STAT signaling pathway”. - To make a connection between the enriched lncRNAs and mRNAs, we screened out the top 20 abun- dant mRNAs. - These data indicate that the PAMs are enriched in immune response-related lncRNAs and their predicted target genes, which allow them to respond quickly to invading organisms.. - Gao et al. - 2a) and the expression of N protein was also confirmed in PRRSV- treated PAMs (Fig. - LncRNA expression profile in PRRSV-infected PAMs Freshly isolated PAMs were treated with PRRSV HuN4 or mock, and followed by RNA-seq methodology. - We next performed GO and KEGG pathway analyses to evaluate the function of DE lncRNAs. - 3d, the upregulated lncRNAs were primarily associ- ated with “NF-κB signaling pathway”, “toll-like receptor signaling pathway”, “MAPK signaling pathway”, “RIG-I- like receptor signaling pathway”, “Jak-STAT signaling pathway”, and “TNF signaling pathway”. - 1 The expression profiles of endogenous lncRNAs and mRNAs in PAMs. - a KEGG pathway analysis of lncRNAs in PAMs. - b Top 20 abundant mRNAs in PAMs. - 3 DE lncRNAs in PAMs upon PRRSV infection. - a Volcano plots of DE lncRNAs between mock and PRRSV-infected PAMs. - Red or green points represent the DE lncRNAs with statistical significance ( P <. - b Unsupervised hierarchical clustering and heat map of lncRNA expression between mock and PRRSV-infected PAMs. - c Verification of lncRNAs expression in mock and PRRSV-infected PAMs.. - The results were presented as the fold change to the corresponding uninfected control. - d and e Pathway analysis of DE lncRNAs after PRRSV infection.. - The color of each dot corresponds to the P -value. - The size of each dot shows the number of lncRNAs. - downregulated lncRNAs were enriched in “PI3K-Akt sig- naling pathway”, “chemokine signaling pathway”, and. - “MAPK signaling pathway” (Fig. - The GO and KEGG pathway patterns suggest that DE lncRNAs are largely associated with the inflammation- and pathogen infection-induced immune responses upon PRRSV infec- tion, indicating that these DE lnRNAs may play critical roles in regulating the virus-induced inflammatory re- sponses in PAMs.. - Gene expression signature in PRRSV-infected PAMs Based on the analysis of RNA-seq data, we also gener- ated volcano plots to visualize the DE profile of protein- coding mRNAs. - Totally 406 mRNAs were differentially expressed (126 upregulated and 280 downregulated mRNAs) in PAMs after PRRSV treatment (Fig. - Figure 4c showed that the upregulated genes were enriched in “TNF signaling pathway”, “cytokine-cytokine receptor interaction”, “che- mokine signaling pathway”, “rheumatoid arthritis”, “Toll- like receptor signaling pathway”, and “inflammatory bowel disease”. - Combined with lncRNA profiles, these results implicate that the DE lncRNAs may have indispensable regulatory functions in the DE genes in PAMs upon virus stimulation.. - Identification of the regulatory function of lncRNAs in immune responses. - Correlation network is an innovative tool to better inte- grate the co-expressed genes with the regulatory func- tions of lncRNAs. - Our results indicate that the DE lncRNAs associated with IFN-inducible genes have the potential to regulate the antiviral functions in macrophages.. - CD163 expression were upregulated, while 22 lncRNAs positively correlated with CD163 expression were downregulated in PRRSV infected cells. - The CD163 module suggests that DE lncRNAs may play import- ant roles in PRRSV entry by regulating the expression level of CD163.. - 4 DE mRNAs in PAMs upon PRRSV infection. - a Volcano plots of mRNAs between mock and PRRSV-infected PAMs. - b Unsupervised hierarchical clustering and heat map of mRNA expression in PAMs upon virus infection. - Additionally, the relationship between these three lncRNAs and their neighboring genes was analyzed based on the genomic location and transcriptome ex- pression profile (Fig. - 5 Correlation between lncRNAs and CXCL2, IFI6/IFITM1, and CD163 mRNA. - The recent growth of next-generation sequencing technology has accelerated the research pro- gress of lncRNAs. - Although tens of thousands of lncRNAs have been discovered, only a small fraction of lncRNAs have been found to regulate gene expression in mouse and human. - However, there are few databases about porcine lncRNAs and their potential functions [35–38], resulting in majority of porcine lncRNAs remains to be unidentified. - We found 2142 lncRNAs abundantly expressed in PAMs. - According to RNA-seq data, we defined a set of 86 DE lncRNAs in PAMs after PRRSV infection. - GO and KEGG pathway enrichment analyses revealed that most DE lncRNAs and protein- coding genes were associated with inflammatory. - 6 Verification of the correlation between lncRNAs and mRNAs. - b The location of lncRNAs and target mRNAs respectively. - pathways in PAMs. - Since the network analysis can pro- vide a global view of all possible lncRNAs-mRNA ex- pression associations based on the different immune background, here, the network analysis was used to pre- dict the functional annotations of DE lncRNAs. - Here, after valid- ation by qPCR, three selected lncRNAs were showed to have a positively correlated expression with their pre- dicted target genes in PAMs. - By using sequence align- ment between full-length lncRNAs and nearby mRNAs, we especially considered protein coding gene loci which act as hosts for lncRNA transcripts. - Only Zeng et al. - These presented data suggest that our proposed method can better reveal the function of lncRNAs not only through the correlation network of different expression but also through cis or trans regulatory mode of lncRNAs located. - Taken together, our findings provide novel insights on functional characterization of lncRNAs in innate im- mune responses of porcine macrophages. - In summary, RNA-seq can be a useful tool to analyze the expression profiles of lncRNAs and their regulatory function. - Here, the transcriptome analysis revealed that a total of 86 DE lncRNAs and 406 DE protein-coding mRNAs were mainly involved in PRRSV-induced inflam- mation and immune responses in PAMs. - Correlation analysis of DE lncRNAs revealed that the predicted target genes of XLOC-022175, XLOC-019295, and XLOC-017089 were related to the inflammation and antiviral signaling pathways. - These DE lncRNAs comprised a valuable atlas that could be used to connect lncRNAs expression with viral pathogenesis. - 200720–01) and experiments were performed according to the regulations and guidelines established by this committee. - Total RNA was extracted from cells with TRIzol (Invi- trogen, SIGMA), and was then reverse transcribed into cDNA using PrimeScript RT reagent Kit with gDNA Eraser by random primers (TaKaRa, Japan) according to the manufacturer’s instructions. - RNA-seq data analysis. - Next, HISAT [60] tools were used to map clean reads to the indexed reference transcriptome. - Function prediction of lncRNAs. - 0.01 and fold change ≥2 were identified as significantly DE lncRNAs using EdgeR. - The potential target genes of DE lncRNAs in cis- and trans- regulatory effects were predicted. - To predict trans target genes, according to the FPKM values of the different expression of lncRNAs and mRNAs in all the samples, the Pearson Correlation coef- ficient between the lncRNAs and the mRNAs were cal- culated, and the threshold for positive correlation was set to Pearson Correlation >. - Differential expression analysis of lncRNAs and mRNAs For each sample, FPKM was a normalized estimation of both mRNA and lncRNA based on RNA-seq data.. - Correlation degrees of lncRNAs and mRNAs were calculated by counting their correlated counter- parts. - PRRSV: Porcine reproductive and respiratory syndrome virus. - Single-cell analysis of long non- coding RNAs in the developing human neocortex. - Porcine reproductive and respiratory syndrome virus (PRRSV): pathogenesis and interaction with the immune system. - Induction of type I interferons by a novel porcine reproductive and respiratory syndrome virus isolate. - Interferon alpha inhibits replication of a live-attenuated porcine reproductive and respiratory syndrome virus vaccine preventing development of an adaptive immune response in swine. - Cytokine expression by macrophages in the lung of pigs infected with the porcine reproductive and respiratory syndrome virus.. - Fetal cytokine response to porcine reproductive and respiratory syndrome virus-2 infection. - Porcine reproductive and respiratory syndrome virus and bacterial endotoxin act in synergy to amplify the inflammatory response of infected macrophages. - Production of porcine TNF α by ADAM17-mediated cleavage negatively regulates porcine reproductive and respiratory syndrome virus infection. - CD163 expression confers susceptibility to porcine reproductive and respiratory syndrome viruses. - Genome-wide analysis of long noncoding RNA and mRNA profiles in PRRSV-infected porcine alveolar macrophages. - Genome-wide analysis of long noncoding RNA profiling in PRRSV-infected PAM cells by RNA sequencing. - Global miRNA, lncRNA, and mRNA Transcriptome profiling of endometrial epithelial cells reveals genes related to porcine reproductive failure caused by porcine reproductive and respiratory syndrome virus. - Transcriptome analysis reveals dynamic Gene expression profiles in porcine alveolar macrophages in response to the Chinese highly pathogenic porcine reproductive and respiratory syndrome virus. - Porcine reproductive and respiratory syndrome virus counteracts the porcine intrinsic virus restriction factors-IFITM1 and Tetherin in MARC-145 cells. - Modulation of CD163 expression by metalloprotease ADAM17 regulates porcine reproductive and respiratory syndrome virus entry. - An attenuated live vaccine based on highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) protects piglets against HP-PRRS
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