- Transcriptome analysis reveals modulation of the STAT family in PEDV-infected IPEC-J2 cells. - Such diseases cause considerable economic losses in the global swine industry. - Enhancing our knowledge of PEDV-induced transcriptomic responses in host cells is imperative to understanding the molecular mechanisms involved in the immune response. - Results: In total, 854 genes were significantly differentially expressed after PEDV infection, including 716 upregulated and 138 downregulated genes. - Functional annotation analysis revealed that the differentially expressed genes were mainly enriched in the influenza A, TNF signaling, inflammatory response, cytokine receptor interaction, and other immune-related pathways. - Next, the putative promoter regions of the 854 differentially expressed genes were examined for the presence of transcription factor binding sites using the MEME tool. - Furthermore, we revealed the regulatory network induced by STAT members in the process of PEDV infection.. - Conclusion: Our transcriptomic analysis described the host genetic response to PEDV infection in detail in IPEC-J2 cells, and suggested that STAT transcription factors may serve as key regulators in the response to PEDV infection.. - These results further our understanding of the pathogenesis of PEDV.. - Porcine epidemic diarrhea (PED), caused by the porcine epidemic diarrhea virus (PEDV), is one of the most severe and globally widespread infectious diseases affecting swine of all ages. - In 2013, PEDV was first reported in the US and spread rapidly nationwide [3]. - 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. - If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. - Full list of author information is available at the end of the article. - PEDV mainly infects and replicates in the villous enterocytes of the small intestine (duodenum, jejunum, and ileum) [5–7], which leads to villous atrophy and vacuolation, as well as a marked reduction in enzymatic activity. - As an important component of the JAK-STAT signaling path- way, STAT proteins are reported to be activated in re- sponse to immunomodulation [17–19]. - After activation, STAT proteins dimerize, translocate to the nucleus, and bind to the promoters of specific target genes, resulting in the regulation of expression of the target genes. - To date, seven mammalian members of the STAT family have been identified, and have been suggested to play different physiological and biological roles.. - In this study, to investigate the host re- sponse patterns to PEDV infection and to identify the key regulators involved in PEDV pathogenesis, the RNA- seq technique was applied to monitor global transcrip- tomic changes in the intestinal porcine epithelial cell line J2 (IPEC-J2). - To further evaluate the response of IPEC-J2 cells to CV777 infection, we then detected the expression of the MX dynamin-like GTPase 1 (MX1) gene, a classic antiviral gene [20]. - Next, the relative transcription level of the CV777 mem- brane protein (M) gene was also calculated. - 1c, a significant increase in the expression level of M gene was detected 48 h after inoculation. - To avoid sample bias, six replicates for each group were collected and used for the construction of the RNA-seq libraries. - Identification of the host gene response to PEDV infection. - The top five genes with the highest changes in the mRNA level were IFIT1, DExD/H-box helicase 58 (DDX58), radical S-adenosyl methionine domain containing 2 (RSAD2), MX2, and forkhead box S1 (FOXS1). - To validate the quality of the RNA-seq data, five differentially expressed genes were random selected and their expression patterns were de- tected by qRT-PCR. - The results showed that the expres- sion patterns of the genes were consistent with the RNA-seq results (Fig. - 3c), although the observed fold changes differed between the qRT-PCR and RNA-seq data, which may reflect differences in the sensitivity and specificity between qRT-PCR and high-throughput se- quencing technology. - Functional analysis of the differentially expressed genes To better understand the functions of the differentially expressed genes, GO and KEGG pathway enrichment analyses were performed. - Among these GO terms were related to the host im- mune response and inflammatory response (Table 2), in- cluding the innate immune response (GO positive regulation of T cell proliferation (GO and regulation of the adaptive immune response (GO:. - KEGG pathway enrichment analysis indicated that the differentially expressed genes were significantly enriched in 41 KEGG pathways (p <. - Interestingly, almost all differentially expressed genes that clustered in these immune-related GO and KEGG pathways were found to be upregulated after CV777 inoculation. - For example, interferon regulatory factor 7 (IRF7), C-C motif chemo- kine ligand 5 (CCL5), STAT1, and interleukin 6 (IL6), which are clustered in the innate immune response (GO:. - In the NF-kappa B signaling pathway, all differentially expressed genes, including CD40 molecule, TRIM25, and nuclear factor kappa B subunit 1 A (NFKB1A), were. - 1 PEDV infection in IPEC-J2 cells. - A significantly higher percentage of apoptotic cells (live (green)/dead (red)) are observed in the infected groups than in the controls (magnification, ×40. - Transcription factor prediction among differentially expressed genes. - The search for significantly overrepresented transcrip- tion factor binding sites in the promoter regions of the differentially expressed genes could be a powerful ap- proach for finding key regulators of complex biological processes. - Therefore, the putative promoter regions (2000 bp upstream of the transcription start site) of the differentially expressed genes were examined for the presence of transcription factor binding sites using the MEME tool. - In particular, five STAT members—STAT1, STAT2, STAT3, STAT4, and STAT5a—were identified as differentially expressed genes by RNA-seq (Fig. - Subsequently, all of the STAT members and their. - As a result promoter sequences for the differentially expressed genes were identified to have at least one STAT1 binding site. - 0.001, Pear- son ’ s Chi-square test) of STAT1 transcription factors among the putative promoters of the differentially expressed genes suggested an important role for STAT factors during the PEDV infection process.. - Construction of a gene regulatory network between the STAT factors and the differentially expressed genes On the basis of the assumption that the STAT protein family-mediated regulatory and signaling networks are Table 1 Descriptive summary of data generated by RNA-Seq. - representative of the infected interactome, STAT factors were used as seeds to construct a gene regulatory net- work. - Figure 5a shows the nodes and interactions at the intersection of the network. - however, it is also necessary to understand the molecular mecha- nisms involved in the host response to PEDV infection.. - Our study, which involved transcriptome analyses, re- vealed 854 significantly differentially expressed genes in the host. - These differentially expressed genes were mainly enriched in the influenza A, TNF signaling path- way, inflammatory response, and other immune-related pathways. - In particular, five members of the STAT. - In the latent state, STAT proteins are inactive as monomers or unpho- sphorylated dimers, which are localized in the cytoplasm of unstimulated target cells [29–32]. - As a non-negligible component of the JAK-STAT signaling pathway, STAT factors play an indispensable role in innate immunity to viral infection. - Each member of the STAT family can be activated by multiple cytokines and their associated JAKs. - STAT1 plays a role in many important cytokine in- duction pathways and can upregulate many pro- inflammatory cytokines, whereas STAT2 is a co-factor in the type I IFN signaling pathway. - 3 Identification of differentially expressed genes. - a Volcano plots of differentially expressed genes between the mock-infected and PEDV-infected groups. - The red and green dots represent upregulated and downregulated genes, respectively, in the PEDV-infected group compared with the mock- infected group. - b Heatmap showing the expression levels of the differentially expressed genes. - c qRT-PCR validation of the differentially expressed genes. - mRNA expression levels were normalized to the mRNA levels of the pig ACTB gene. - PEDV infection. - reported the distinct and potentially opposing roles of STAT5a and STAT5b in the regulation of hepatic drug response genes [41].. - STAT5a is expressed at a much lower level in the liver than STAT5b [42]. - In the present study, our findings revealed for the first time that Table 2 The GO terms were related to host immune response and inflammatory response. - a Enrichment of putative transcription factor binding motifs in the promoter regions of differentially expressed genes detected by MEME. - The right panel shows the annotation of the enriched motifs as determined by MAST (ID, number, p value). - a PPI network of the differentially expressed genes based on STRING analysis. - The hub genes of the network are shown in yellow. - STAT5a, but not STAT5b, regulated the expression of key factors involved in the immune response after virus infection.. - For ex- ample, nearly all of the detected IRFs were reported to be downregulated after PEDV infection in Vero E6 cells [44], whereas our results showed that IRF1, FIRF2, IRF7, and IRF9 were significantly upregulated after PEDV in- fection in IPEC-J2 cells, and other detected IRF mem- bers were not significantly changed. - A recent study performed global mapping of H3K4 trimethylation and transcriptomic analyses in the PEDV- infected jejunum of piglets compared with healthy pig- lets, using chromatin immunoprecipitation sequencing and RNA-seq, and provided novel insights into our un- derstanding of the host response to PEDV infection [14].. - In particular, IRF3 and IRF7 are dir- ect transducers of virus-mediated signaling in the induc- tion of type I IFN [50]. - Unexpectedly, phosphoinositide-3-kinase adaptor protein 1 (PIK3AP1), a member of the vital PIKs, was significantly upregulated during PEDV infec- tion at the transcriptional level. - PEDV infection of IPEC-J2 cells. - Cells were then incubated in a mixture of 1 μL of 1000× Calcein-AM solution, 1 μL of 1000× propidium iodide (PI) working solution, and 500 μL of PBS for 15 min at 37 °C in the dark. - A single nucleotide A (adenine) was added (A-tailing) to the 3′ end of the end-repaired DNA fragments. - The minimal number of PCR cycles (15 cycles) was used to avoid skewing the representation of the library.. - The Cuffdiff software was used to screen the differentially expressed genes [53. - Functional enrichment of the gene module was analyzed using the web-based tools in DAVID (v6.8. - We interrogated enrichments of transcription factors in the promoters of differentially expressed genes using the MEME Suite (http://meme-suite.org/index.html). - First, the putative promoter regions (2000 bp upstream of the transcription start site) of the differentially expressed genes were extracted using the BioMart program in the Ensembl database. - In the network, each node represents a protein and each edge represents an interaction between two proteins.. - Statistical analyses of the qRT-PCR results were carried out using SPSS 20.0 (IBM Corp., Armonk, NY, USA).. - Information on each significantly differentially expressed gene ( p <. - GO terms significantly enriched among the differentially expressed genes in PEDV-infected cells.. - KEGG pathways significantly enriched among the differentially expressed genes in PEDV- infected cells.. - JL, JR, XZ and KW revised of the manuscript. - The datasets generated during the current study are available in the NCBI Sequence Read Archive under the accession number PRJNA599091.. - Three- dimensional sequential study of the intestinal surface in experimental porcine CV 777 coronavirus enteritis. - Identification and comparison of receptor binding characteristics of the spike protein of two porcine epidemic diarrhea virus strains. - Contribution of the porcine aminopeptidase N (CD13) receptor density to porcine epidemic diarrhea virus infection. - Global mapping of H3K4 Trimethylation (H3K4me3) and Transcriptome analysis reveal genes involved in the response to epidemic diarrhea virus infections in pigs. - The interleukin-6-activated acute-phase response factor is antigenically and functionally related to members of the signal transducer and activator of transcription (STAT) family. - In silico structure analysis of alphaviral RNA genomes shows diversity in the evasion of IFIT1-mediated innate immunity. - Isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the United States. - STATs Dimerize in the absence of phosphorylation. - Genome-wide analysis of differentially expressed genes and the modulation of PEDV infection in Vero E6 cells. - A unifying view of the broad-spectrum antiviral activity of RSAD2 (viperin) based on its radical-SAM chemistry. - Viral exploitation of the MEK/ERK pathway - a tale of vaccinia virus and other viruses. - accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions
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