Xinyun Li

Understanding Haemophilus parasuis infection in porcine spleen through a transcriptomics approach.

BackgroundHaemophilus parasuis (HPS) is an important swine pathogen that causes Glässers disease, which is characterized by fibrinous polyserositis, meningitis and arthritis. The molecular mechanisms that underlie the pathogenesis of the disease remain poorly understood, particularly the resistance of porcine immune system to HPS invasion. In this study, we investigated the global changes in gene expression in the spleen following HPS infection using the Affymetrix Porcine Genechip™.ResultsA total of 931 differentially expressed (DE) transcripts were identified in the porcine spleen 7 days after HPS infection; of these, 92 unique genes showed differential expression patterns based on analysis using BLASTX and Gene Ontology. The DE genes involved in the immune response included genes for inflammasomes (RETN, S100A8, S100A9, S100A12), adhesion molecules (CLDN3, CSPG2, CD44, LGALS8), transcription factors (ZBTB16, SLC39A14, CEBPD, CEBPB), acute-phase proteins and complement (SAA1, LTF, HP, C3), differentiation genes for epithelial cells and keratinocytes (TGM1, MS4A8B, CSTA), and genes related to antigen processing and presentation (HLA-B, HLA-DRB1). Further immunostimulation analyses indicated that mRNA levels of S100A8, S100A9, and S100A12 in porcine PK-15 cells increased within 48 h and were sustained after administration of lipopolysaccharide (LPS) and Poly(I:C) respectively. In addition, mapping of DE genes to porcine health traits QTL regions showed that 70 genes were distributed in 7 different known porcine QTL regions. Finally, 10 DE genes were validated by quantitative PCR.ConclusionOur findings demonstrate previously unrecognized changes in gene transcription that are associated with HPS infection in vivo, and many potential cascades identified in the study clearly merit further investigation. Our data provide new clues to the nature of the immune response in mammals, and we have identified candidate genes that are related to resistance to HPS.

Inhibition of miR-214 expression represses proliferation and differentiation of C2C12 myoblasts

MicroRNAs (miRNAs) are small non‐coding RNAs that participate in diverse biological processes including skeletal muscle development. MiR‐214 is an miRNA that is differentially expressed in porcine embryonic muscle and adult skeletal muscle, suggesting that miR‐214 may be related to embryonic myogenesis. In this study, the myoblast cell line C2C12 was used for functional analysis of miR‐214 in vitro. The results showed that miR‐214 was expressed both in myoblasts and in myotubes and was upregulated during differentiation. After treatment with an miR‐214 inhibitor and culturing in differentiation medium, myoblast differentiation was repressed, as indicated by the significant downregulation of expression of the myogenic markers myogenin and myosin heavy chain (MyHC). Interestingly, myoblast proliferation was also repressed when cells were transfected with an miR‐214 inhibitor and cultured in growth medium by real‐time proliferation assay and cell cycle analysis. Our results showed that miR‐214 regulates both proliferation and differentiation of myoblasts depending on the conditions. Copyright

Transcriptome analysis of mRNA and miRNA in skeletal muscle indicates an important network for differential Residual Feed Intake in pigs

Feed efficiency (FE) can be measured by feed conversion ratio (FCR) or residual feed intake (RFI). In this study, we measured the FE related phenotypes of 236 castrated purebred Yorkshire boars, and selected 10 extreme individuals with high and low RFI for transcriptome analysis. We used RNA-seq analyses to determine the differential expression of genes and miRNAs in skeletal muscle. There were 99 differentially expressed genes identified (q ≤ 0.05). The down-regulated genes were mainly involved in mitochondrial energy metabolism, including FABP3, RCAN, PPARGC1 (PGC-1A), HK2 and PRKAG2. The up-regulated genes were mainly involved in skeletal muscle differentiation and proliferation, including IGF2, PDE7A, CEBPD, PIK3R1 and MYH6. Moreover, 15 differentially expressed miRNAs (|log2FC| ≥ 1, total reads count ≥ 20, p ≤ 0.05) were identified. Among them, miR-136, miR-30e-5p, miR-1, miR-208b, miR-199a, miR-101 and miR-29c were up-regulated, while miR-215, miR-365-5p, miR-486, miR-1271, miR-145, miR-99b, miR-191 and miR-10b were down-regulated in low RFI pigs. We conclude that decreasing mitochondrial energy metabolism, possibly through AMPK - PGC-1A pathways, and increasing muscle growth, through IGF-1/2 and TGF-β signaling pathways, are potential strategies for the improvement of FE in pigs (and possibly other livestock). This study provides new insights into the molecular mechanisms that determine RFI and FE in pigs.

Identification of microRNAs involved in dexamethasone-induced muscle atrophy

MicroRNAs (miRNAs), a novel class of post-transcriptional gene regulators, have been demonstrated to be involved in several cellular processes regulating the expression of protein-coding genes. To investigate the mechanisms of miRNA-mediated regulation during the process of muscle atrophy, we performed miRNA microarray hybridization between normal differentiated C2C12 cells and dexamethasone (DEX)-treated C2C12 cells. We observed that 11 miRNAs were significantly up-regulated and six miRNAs were down-regulated in the differentiated C2C12 cells after being treated with DEX. Stem–loop real-time RT-PCR confirmed the differential expression of six selected miRNAs (miR-1, miR-147, miR-322, miR-351, and miR-503*, miR-708). miRNA potential target prediction was accomplished using TargetScan, and many target genes related to muscle growth and atrophy have been reported in previous studies. The results of the current study suggested the potential roles of these differentially expressed miRNAs in skeletal muscle atrophy.

Japanese encephalitis virus infects porcine kidney epithelial PK15 cells via clathrin- and cholesterol-dependent endocytosis.

BackgroundJapanese encephalitis virus (JEV) is a mosquito-borne flavivirus that causes acute viral encephalitis in humans. Pigs are important amplifiers of JEV. The entry mechanism of JEV into porcine cells remains largely unknown. In this study, we present a study of the internalization mechanism of JEV in porcine kidney epithelial PK15 cells.ResultsWe demonstrated that the disruption of the lipid raft by cholesterol depletion with methyl-β-cyclodextrin (MβCD) reduced JEV infection. We also found that the knockdown of clathrin by small interfering RNA (siRNA) significantly reduced JEV-infected cells and JEV E-glycoprotein levels, suggesting that JEV utilizes clathrin-dependent endocytosis. In contrast, the knockdown of caveolin-1, a principal component of caveolae, had only a small (although statistically significant) effect on JEV infection, however, JEV entry was not affected by genistein. These results suggested that JEV entry was independent of caveolae.ConclusionsTaken together, our results demonstrate that JEV enters porcine kidney epithelial PK15 cells through cholesterol- and clathrin-mediated endocytosis.

Pseudorabies viral replication is inhibited by a novel target of miR-21.

The pseudorabies virus (PRV) is a porcine virus classified as a member of the Alphaherpesvirinae subfamily of Herpesviridae. Recent studies have confirmed that viruses regulate the gene expression in host cells. Commonly affected genes include oxidative-stress response genes, genes involved in the phosphatidylinositol 3-kinase/Protein Kinase B (PI3K/Akt) signaling pathway, and interferon- and interleukin-related genes. However, the post-transcriptional regulation of host genes following PRV infection is hitherto unclear. In this study, we used miRNA microarray approaches to assess miRNA expression in PRV-infected porcine kidney 15 cell line (PK-15), and observed that miR-21 was expressed at high level 12h after the cells were infected with PRV. Furthermore, we identified chemokine (C-X-C motif) ligand 10 (CXCL10), also named interferon-γ inducible protein-10 (IP-10), as a novel target gene of miR-21. IP-10 was down-regulated at 4h after PRV infection. PRV replication was significantly inhibited by IP-10 overexpression.

Molecular Characterization of Caveolin-1 in Pigs Infected with Haemophilus parasuis

Caveolin-1 (Cav1) plays a critical role in the invasion of pathogenic microbes into host cells, yet little is known about porcine Cav1. In this study, we provide the molecular characterization of Cav1 in pigs following stimulation with LPS/polyinosinic-polycytidylic acid as well as during infection with Haemophilus parasuis. The porcine Cav1 gene is 35 kb long and is located at SSC18q21; two isoforms (Cav1-α and Cav1-β) are produced by alternative splicing. Three point mutations were identified in the coding region of the gene, two of which were significantly associated with nine immunological parameters in Landrace pigs, including the Ab response against porcine reproductive and respiratory syndrome virus and lymphocyte counts. Promoter analysis indicated that NF-κB activates both Cav1 transcripts, but the forkhead gene family specifically regulates Cav1-β in the pig. Porcine Cav1 is expressed ubiquitously, with Cav1-α more abundantly expressed than Cav1-β in all tissues investigated. Basal expression levels of Cav1 in PBMCs are relatively similar across different pig breeds. LPS and polyinosinic-polycytidylic acid markedly induced the expression of Cav1 in porcine kidney-15 cells in vitro, likely through NF-κB activation. Pigs infected with H. parasuis exhibited decreased expression of Cav1, particularly in seriously impaired organs such as the brain. This study provides new evidence that supports the use of Cav1 as a potential diagnostic and genetic marker for disease resistance in animal breeding. In addition, our results suggest that Cav1 may be implicated in the pathogenesis of Glasser’s disease, which is caused by H. parasuis.

Molecular characterisation of porcine miR-155 and its regulatory roles in the TLR3/TLR4 pathways

MiR-155 plays very important roles in host inflammation and immunity. However, few studies have focused on miR-155 in livestock. In this study, the molecular characterisation of miR-155 and its functional roles in TLR3/TLR4 signalling pathways were investigated in pigs. The results indicated that miR-155 was highly expressed in the spleen and fat tissues of the pig. In PK-15 cells, miR-155 was up-regulated 4h after LPS stimulation and up-regulated 12h and 24h after poly (I:C) stimulation. Furthermore, the overexpression of miR-155 significantly activated the TLR3/TLR4 signalling pathways, and the inhibition of miR-155 suppressed these pathways. Thus, miR-155 played positive regulatory roles in TLR3/TLR4 signalling pathways. Additionally, one T/C SNP of miR-155 was significantly associated with basophil percentage (BA%), absolute eosinophili value (EO) and the distribution width of the least squares mean of CD3-CD4-CD8+ T cells (DWT) in pigs. Our study offers new evidence on the immune function of miR-155 in pigs.

Characterization, expression profiles, intracellular distribution and association analysis of porcine PNAS-4 gene with production traits.

BackgroundIn a previous screen to identify differentially expressed genes associated with embryonic development, the porcine PNAS-4 gene had been found. Considering differentially expressed genes in early stages of muscle development are potential candidate genes to improve meat quality and production efficiency, we determined how porcine PNAS-4 gene regulates meat production. Therefore, this gene has been sequenced, expression analyzed and associated with meat production traits.ResultsWe cloned the full-length cDNA of porcine PNAS-4 gene encoding a protein of 194 amino acids which was expressed in the Golgi complex. This gene was mapped to chromosome 10, q11–16, in a region of conserved synteny with human chromosome 1 where the human homologous gene was localized. Real-time PCR revealed that PNAS-4 mRNA was widely expressed with highest expression levels in skeletal muscle followed by lymph, liver and other tissues, and showed a down-regulated expression pattern during prenatal development while a up-regulated expression pattern after weaning. Association analysis revealed that allele C of SNP A1813C was prevalent in Chinese indigenous breeds whereas A was dominant allele in Landrace and Large White, and the pigs with homozygous CC had a higher fat content than those of the pigs with other genotypes (P < 0.05).ConclusionPorcine PNAS-4 protein tagged with green fluorescent protein accumulated in the Golgi complex, and its mRNA showed a widespread expression across many tissues and organs in pigs. It may be an important factor affecting the meat production efficiency, because its down-regulated expression pattern during early embryogenesis suggests involvement in increase of muscle fiber number. In addition, the SNP A1813C associated with fat traits might be a genetic marker for molecular-assisted selection in animal breeding.

Porcine S100A8 and S100A9: Molecular characterizations and crucial functions in response to Haemophilus parasuis infection

S100 calcium-binding protein A8 (S100A8) and S100 calcium-binding protein A9 (S100A9) are pivotal mediators of inflammatory and protective anti-infection responses for the mammalian host. In this study, we present the molecular cloning of porcine S100A8 (pS100A8) and porcine S100A9 (pS100A9). Both genes comprise 3 exons and 2 introns and are located on pig chromosome 4q21-q23 (closely linked to SW512). Homology comparison to other mammalian species affirmed that critical functional amino acids for post-transcriptional modification, inflammatory regulation, and formation of heterodimeric complexes exist in pS100A8 and pS100A9. Under normal conditions, both genes are preferentially expressed in porcine immune or immune-related organs, e.g., bone marrow, spleen, lymph nodes, and lung. Upon stimulation in porcine whole blood cultures with LPS or Poly(I:C), they are dramatically induced. Interestingly, the maximum increase of mRNA levels in blood cultures of Meishan pigs is significantly greater than that in Duroc pigs. We previously showed that pS100A8 and pS100A9 mRNA were up-regulated following Haemophilus parasuis (HPS) infection. We herein further confirm their up-regulation at the protein level in multiple HPS infected tissues (spleen, lung and liver). Functional cluster and network analysis based on our previous microarray data discovered that CEBPB may be one of the key transcription factors. A pS100A8/pS100A9-CASP3-SLC1A2 pathway regulating lipid metabolism was found. Both of their pro- and anti-inflammatory functions in response to HPS infection are highlighted.