Mengjin Zhu

LongSAGE analysis of skeletal muscle at three prenatal stages in Tongcheng and Landrace pigs

BackgroundObese and lean pig breeds show obvious differences in muscle growth; however, the molecular mechanism underlying phenotype variation remains unknown. Prenatal muscle development programs postnatal performance. Here, we describe a genome-wide analysis of differences in prenatal skeletal muscle between Tongcheng (a typical indigenous Chinese breed) and Landrace (a leaner Western breed) pigs.ResultsWe generated transcriptome profiles of skeletal muscle from Tongcheng and Landrace pigs at 33, 65 and 90 days post coitus (dpc), using long serial analysis of gene expression (LongSAGE). We sequenced 317,115 LongSAGE tags and identified 1,400 and 1,201 differentially expressed transcripts during myogenesis in Tongcheng and Landrace pigs, respectively. From these, the Gene Ontology processes and expression patterns of these differentially expressed genes were constructed. Most of the genes showed different expression patterns in the two breeds. We also identified 532, 653 and 459 transcripts at 33, 65 and 90 dpc, respectively, that were differentially expressed between the two breeds. Growth factors, anti-apoptotic factors and genes involved in the regulation of protein synthesis were up-regulated in Landrace pigs. Finally, 12 differentially expressed genes were validated by quantitative PCR.ConclusionOur data show that gene expression phenotypes differ significantly between the two breeds. In particular, a slower muscle growth rate and more complicated molecular changes were found in Tongcheng pigs, while genes responsible for increased cellular growth and myoblast survival were up-regulated in Landrace pigs. Our analyses will assist in the identification of candidate genes for meat production traits and elucidation of the development of prenatal skeletal muscle in mammals.

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.

Identification and characterization of microRNAs from porcine skeletal muscle

MicroRNAs (miRNAs) are a class of non-coding RNAs that negatively regulate gene expression at the post-transcriptional level. There is increasing evidence to suggest that miRNAs participate in muscle development in mice and humans; however, few studies have focused on miRNAs in porcine muscle tissue. Here, we experimentally detected and identified conserved and unique miRNAs from porcine skeletal muscle. Fifty-seven distinct miRNAs were identified, of which 39 have not been reported earlier in the pig. Of these, two miRNAs appear to be novel and pig-specific. Surprisingly, these two differ only by a single nucleotide. A part of their primary transcript was cloned and confirmed by sequencing analysis. Alignment of the two sequences using ClustalW showed that the precursor sequences were almost identical, but the flanking sequences were different, indicating that these two novel miRNAs may represent rapidly evolving miRNAs in the pig genome. The expression patterns of eight miRNAs were characterized by real-time polymerase chain reaction of eight pig tissue samples. The ssc-let-7e and ssc-miR-181b miRNAs were expressed in all tissues analysed. The ssc-let-7c, ssc-miR-125b, ssc-miR-new1 and ssc-miR-new2 miRNAs were expressed in several tissues, while ssc-miR-122 and ssc-miR-206 were specifically expressed in the liver and muscle respectively. Our results add to existing data on porcine miRNAs and are useful for investigating the biological functions of miRNAs in porcine skeletal muscle development.

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.

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.

Immunogenomics for identification of disease resistance genes in pigs: a review focusing on Gram-negative bacilli

Over the past years, infectious disease has caused enormous economic loss in pig industry. Among the pathogens, gram negative bacteria not only cause inflammation, but also cause different diseases and make the pigs more susceptible to virus infection. Vaccination, medication and elimination of sick pigs are major strategies of controlling disease. Genetic methods, such as selection of disease resistance in the pig, have not been widely used. Recently, the completion of the porcine whole genome sequencing has provided powerful tools to identify the genome regions that harboring genes controlling disease or immunity. Immunogenomics, which combines DNA variations, transcriptome, immune response, and QTL mapping data to illustrate the interactions between pathogen and host immune system, will be an effective genomics tool for identification of disease resistance genes in pigs. These genes will be potential targets for disease resistance in breeding programs. This paper reviewed the progress of disease resistance study in the pig focusing on Gram-negative bacilli. Major porcine Gram-negative bacilli and diseases, suggested candidate genes/pathways against porcine Gram-negative bacilli, and distributions of QTLs for immune capacity on pig chromosomes were summarized. Some tools for immunogenomics research were described. We conclude that integration of sequencing, whole genome associations, functional genomics studies, and immune response information is necessary to illustrate molecular mechanisms and key genes in disease resistance.

Whole blood transcriptome comparison of pigs with extreme production of in vivo dsRNA-induced serum IFN-a

Interferon (IFN) is one of the major regulators of innate immunity, it also mediates the adaptive immune responses to a broad spectrum of pathogens. This study aims in identifying differences between high vs. low INF-a responders which were chosen based on serum INF-a levels at 4 h post poly I:C treatment. A transcriptomic analysis was designed to describe the whole blood differential transcriptomal response to poly I:C by pigs with high vs. low IFN alpha levels. The capability of producing dsRNA (poly I:C)-induced serum IFN-a is highly variable in pig population. The high INF-a responders had 328 unique differentially expressed genes, suggesting that the HIGH pigs have greater responsiveness upon the dsRNA simulation. Based on the results, the interferon-dependent antiviral responsiveness through the IFN-stimulated genes (ISGs) is likely more effective in HIGH pigs. Inferring from the known organization of IFN pathways, the reason for the more IFN-a production in the HIGH pigs was likely due to the enhanced expression of IRF-7 in TLR or RIG- I/MDA5 signaling pathways. Furthermore, the larger number of the altered genes in the HIGH pigs after simulation is also possibly because of the greater number of the altered transcription factors. To our knowledge, this is the first report of comparative transcriptomic analysis to advance our understanding of whole blood immune response in pigs with different in vivo poly I:C-inducted IFN-a levels. The paper significantly expands our knowledge of how pigs respond to poly I:C which is highly relevant for understanding resistance to viral infections and also for vaccine development.

Systems infection biology: a compartmentalized immune network of pig spleen challenged with Haemophilus parasuis

BackgroundNetwork biology (systems biology) approaches are useful tools for elucidating the host infection processes that often accompany complex immune networks. Although many studies have recently focused on Haemophilus parasuis, a model of Gram-negative bacterium, little attention has been paid to the hosts immune response to infection. In this article, we use network biology to investigate infection with Haemophilus parasuis in an in vivo pig model.ResultsBy targeting the spleen immunogenome, we established an expression signature indicative of H. parasuis infection using a PCA/GSEA combined method. We reconstructed the immune network and estimated the network topology parameters that characterize the immunogene expressions in response to H. parasuis infection. The results showed that the immune network of H. parasuis infection is compartmentalized (not globally linked). Statistical analysis revealed that the reconstructed network is scale-free but not small-world. Based on the quantitative topological prioritization, we inferred that the C1R-centered clique might play a vital role in responding to H. parasuis infection.ConclusionsHere, we provide the first report of reconstruction of the immune network in H. parasuis-infected porcine spleen. The distinguishing feature of our work is the focus on utilizing the immunogenome for a network biology-oriented analysis. Our findings complement and extend the frontiers of knowledge of host infection biology for H. parasuis and also provide a new clue for systems infection biology of Gram-negative bacilli in mammals.