- Differentially expressed non-coding RNAs induced by transmissible gastroenteritis virus potentially regulate inflammation and NF- κ B pathway in porcine intestinal. - A total of 523 mRNAs, 65 microRNAs (miRNAs), and 123 circular RNAs (circRNAs) were differentially expressed. - Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed differentially expressed mRNAs were linked to inflammation-related pathways, including NF- κ B, Toll-like receptor, NOD-like receptor, Jak-STAT, TNF, and RIG-I-like receptor pathways. - The data showed that ssc_circ_009380 and miR-22 might have interaction relationship. - Dual-luciferase reporter assay confirmed that miR-22 directly bound to ssc_circ_009380. - We also observed that overexpression of miR-22 led to a reduction of p-I κ B- α and accumulation of p65 in nucleus in TGEV- infected IPEC-J2 cells. - In contrast, inhibition of miR-22 had the opposite effects. - Moreover, silencing of ssc_circ_009380 inhibited accumulation of p65 in nucleus and phosphorylation of I κ B- α. - Conclusions: The data revealed that differentially expressed mRNAs and ncRNAs were primarily enriched in. - inflammation-related pathways and ssc_circ_009380 promoted activation of NF- κ B pathway by binding miR-22 during TGEV-induced inflammation.. - We previously reported that TGEV infection led to 21 differentially expressed miRNAs, among which miR-4331 inhibited transcription of TGEV gene7 via targeting CDCA7 [16], and miR-27b functioned as a negative regulator during TGEV-induced apoptosis [17]. - ducted bioinformatics analysis of differentially expressed mRNAs, miRNAs, and circRNAs. - ssc_circ_009380 attenuated TGEV-induced activation of NF- κ B pathway via binding miR-22.. - TGEV infection induces inflammatory response in IPEC-J2 IPEC-J2 cells were infected with TGEV at 1.0 MOI for 12 h, 24 h, and 36 h. - Sequencing and analysis of ncRNAs and mRNAs of TGEV- infected IPEC-J2. - In total, 523 differentially expressed mRNAs (in- cluding 462 up-regulated mRNAs and 61 down-regulated mRNAs), 65 differentially expressed miRNAs (including 46 up-regulated miRNAs and 19 down-regulated miRNAs) and 123 differentially expressed circRNAs (including 69 up- regulated circRNAs and 54 down-regulated circRNAs) were obtained (Fig. - In addition, there were 45 novel miRNAs in the differentially expressed miRNAs. - KEGG analysis of differentially expressed miRNAs, circRNAs, and mRNAs. - We predicted the targets of differentially expressed miRNAs and source genes of differentially expressed circRNAs. - a The relative mRNA level of IL-8 in IPEC-J2 infected with TGEV for 0 h, 12 h, 24 h, or 36 h. - b The relative mRNA level of IL-6 in IPEC-J2 infected with TGEV for 0 h, 12 h, 24 h, or 36 h. - c The relative mRNA level of TNF- α in IPEC-J2 infected with TGEV for 0 h, 12 h, 24 h, or 36 h. - genes and differentially expressed mRNAs were respectively searched for functional enrichments by a KEGG database search (http://www.genome.jp/kegg. - KEGG analysis results indicated that differentially expressed mRNAs were mostly involved in Toll-like receptor, RIG-I-like receptor, TNF, NOD-like receptor, Jak-STAT, and NF-κB pathways (Fig. - Differentially expressed miRNAs were primarily enriched in B cell receptor and Toll-like receptor pathways (Fig. - The source genes of differentially expressed circRNAs were in- volved in RIG-I-like receptor, TNF, NOD-like receptor, and NF-κB pathways (Fig. - To predict the interaction between ncRNAs and mRNAs during TGEV infection, the intersection of differentially expressed targets of miRNAs and mRNAs were collected.. - The results showed that ssc_circ_001964, ssc_circ_000470, ssc_circ_005884, and ssc_circ_009380 are circular (Fig. - To validate the reliability of RNA sequencing, the expression levels of differentially expressed miRNAs (miR-218, mir-2413, mir-492, mir-7550, mir-2510, and miR-22), mRNAs (DDX58, CCL5, and IL-6), and cir- cRNAs (ssc_circ_001964, ssc_circ_000470, ssc_circ_. - 005884, and ssc_circ_009380) were measured by qRT- PCR. - miR-22 directly binds to ssc_circ_009380. - In the present study, we demonstrated that TGEV infection activated inflammation-related pathways and up-regulated miR-22. - It was reported that miR-22 was involved in. - 2 Clustering and Heatmap analysis of differentially expressed mRNAs (FPKM), miRNAs (TPM), and circRNAs (RPKM) across TGEV infection (T1, T2) and Mock infection (M1, M2). - a Clustering and Heatmap analysis of differentially expressed mRNAs, including 462 up-regulated mRNAs and 61 down-regulated mRNAs. - b Clustering and Heatmap analysis of differentially expressed miRNAs, including 46 up-regulated miRNAs and 19 down- regulated miRNAs. - c Clustering and Heatmap analysis of differentially expressed circRNAs, including 69 up-regulated circRNAs and 54 down- regulated circRNAs. - Therefore, we speculated that miR-22 might play a role in TGEV-induced inflammation response. - Based on bioinfor- matics analysis, miR-22 might bind to ssc_circ_009380. - To confirm that, the sequence of ssc_circ_009380 was ampli- fied by PCR and cloned into 3 ′ UTR of Renilla luciferase in psiCHECK-2 to construct wild-type (WT) recombinant plasmid psi-ssc_circ_009380-WT. - miR-22 in ssc_circ_009380 were mutated by point mutation to construct mutant recombinant plasmid psi-ssc_circ_. - The recombinant plasmids were respectively co-transfected into IPEC-J2 cells together with miR-22 mimics (or miRNA mimics control, miR-22 inhibitors, miRNA inhibitors control). - miR-22 was overex- pressed using miR-22 mimics and inhibited using miR-22 in- hibitors (Fig. - The luciferase activities of psi-ssc_circ_009380-WT was decreased at 62% by miR-22 mimics and not affected by miR-22 inhibitors (Fig. - However, the luciferase activities of psi-ssc_ci rc_009380-Mut was not affected by miR-22 mimics and inhibitors (Fig. - 7e and f ).The effects of miR-22 and ssc_circ_009380 on TGEV-induced activation of NF-κB in IPEC-J2.. - To evaluate the effect of miR-22 on TGEV-induced NF-κB activation, IPEC-J2 cells were transfected with miR-22 mimics (or miRNA mimics control, miR-22 inhib- itors, miRNA inhibitors control) and subsequently in- fected with TGEV at 1 MOI for 24 h. - The miR-22 was overexpressed by miR-22 mimics and inhibited by miR-22 inhibitors (Fig. - p-IκB-α and nucleic distribution of p65 were suppressed by miR-22 mimics and increased by miR-22 inhibitors, but not affected by miR-22 mimics and inhibitors in cytoplasm (Fig. - To evaluate the effect of ssc_circ_009380 on TGEV- induced activation of NF-κB, IPEC-J2 cells were trans- fected with siRNA of ssc_circ_009380 (siCirc009380) (or negative control) and subsequently infected with TGEV at 1 MOI for 24 h. - The ssc_circ_009380 level was down-regulated by siCirc009380 (Fig. - In the present study, differentially expressed 523 mRNAs, 65 miRNAs, and 123 circRNAs were obtained. - KEGG analysis showed that differentially expressed mRNAs and ncRNAs were primarily enriched in inflammation-related pathways. - circRNA ssc_circ_009380 was identified as the sponge of miR-22 and enhanced activation of NF-κB path- way through binding miR-22 during TGEV infection.. - In this study, the 523 differentially expressed mRNAs are enriched in Toll-like receptors, RIG-I-like receptors, TNF, NOD-like receptors, Jak-STAT, and NF-κB signaling pathways.. - 3 KEGG analysis of differentially expressed mRNAs and ncRNAs.. - a KEGG enrichment analysis of differentially expressed mRNAs. - b Target genes of differentially expressed miRNAs. - c Source genes of differentially expressed circRNAs. - Consistently, we find TGEV infection upregulates RIG-I and TLR3 and induces the activation of NF-κB pathway in IPEC-J2 cells, indicating TGEV. - a Venn diagram shows the number of overlap genes in target genes of differentially expressed miRNAs. - study, TGEV infection activates the Jak-STAT signaling pathway in IPEC-J2 cells, consistent with previous studies.. - In this study, miRNAs profile and circRNAs profile were changed during TGEV infection in IPEC-J2 cell line. - miR-22 is involved in inhib- ition of myocardial ischemia-reperfusion injury by an anti-inflammation mechanism [26]. - We demonstrated that miR-22 was up-regulated by TGEV infection and had an anti-inflammation effect by targeting IL-6 and restraining DDX58, and CCL5, which is correlated with previous reports, but through different mechanisms. - We found that miR-22 could be directly attached to ssc_circ_9380 and mediate TGEV-induced inflammation response, suggesting ssc_circ_9380 acts as the sponge of miR-22.. - In the present study, we performed the next generation sequencing to obtain the profiles of mRNAs, miRNAs, and circRNAs of IPEC-J2 cells during TGEV infection and investigated the effects of differentially expressed. - b The relative levels of differentially expressed mRNAs, miRNAs, and circRNAs. - The data reveal that the differentially expressed mRNAs and ncRNAs are enriched in inflammation-related pathways and ssc_circ_009380 potentiate TGEV-induced activa- tion of NF-κB pathway through attaching to miR-22.. - IPEC-J2 cell line was kindly gifted by Dr. - miR-22 mimics, miRNA mimics control, miR-22 inhibitors, miRNA inhibi- tors control, siCirc009380, and negative control were syn- thesized by GenePharma (China) (The sequences were shown in Additional file 1: Table S8).. - IPEC-J2 cells were infected with TGEV at 1 MOI for 24 h (indicated by T1 and T2). - 7 Directly binding of miR-22 to ssc_circ_009380. - The relative level of miR-22 in IPEC-J2 treated with (a) Schematic overview of dual-luciferase report plasmid. - The upper sequences are the binding sites of miR-22. - The middle sequences are the sequences of mature miR- 22. - The lower sequences are the mutated miR-22 binding sites. - c miR-22 mimics and d miR-22inhibitors. - e and f The relative luciferase activities of psi-ssc_circ_009380-WT and psi-ssc_circ_009380-Mut in response to miR-22 mimics and miR-22 inhibitors. - 8 The effects of miR-22 and ssc_circ_009380 on TGEV-induced activation of NF- κ B pathway in IPEC-J2. - a Overexpression effect of miR-22 mimics. - b Knockdown effect of miR-22 inhibitors. - c Knockdown effect of siCirc009380 on ssc_circ_009380. - The relative levels of miR-22 and ssc_circ_009380 were measured by real-time PCR (normalized to U6 and in reference to the control. - d, e, and f The effects of miR-22 and ssc_circ_009380 on p65, I κ B- α , p- I κ B- α. - According to the genome positions and hairpin struc- tures predicted by software Mireap (v0.2), the novel miRNA candidates were obaineds.. - Significance analysis of miRNAs, mRNAs, and circRNAs The edgeR package (http://www.r-project.org/) was used to identify differentially expressed miRNAs, mRNAs, and cir- cRNAs. - 0.05 were set as thresholds for significant differentially expressed miR- NAs and circRNAs. - KEGG and interaction analysis of differentially expressed mRNAs, miRNAs, and circRNAs. - The binding sites of miR-22 in targets and circRNA were mutated [16]. - IPEC-J2 cells were co-transfected with 100 ng of plasmid and 100 nM of miR-22 mimics (or miRNA mimics control, miR-22 in- hibitors, miRNA inhibitors control) using Lipofecta- mine 3000 (Invitrogen, US). - The detailed information of differentially expressed mRNAs. - The detailed information of differentially expressed miRNAs. - The detailed information of differentially expressed circRNAs. - The target genes of differentially expressed miRNAs. - Source genes of differentially expressed circRNAs. - The sequences of miR-22 mimics and inhibitors. - IPEC- J2: Porcine intestinal epithelial cells-jejunum 2. - Extracellular Uridine triphosphate and adenosine triphosphate attenuate endothelial inflammation through miR-22-mediated ICAM-1 inhibition. - Microarray analysis of differentially expressed transcripts in porcine intestinal epithelial cells (IPEC- J2) infected with porcine sapelovirus as a model to study innate immune responses to enteric viruses
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