Comparative Biochemistry and Physiology Part D: Genomics and Proteomics
Identification of differentially expressed miRNAs through high-throughput sequencing in the chicken lung in response to Mycoplasma gallisepticum HS
Introduction
Mycoplasmosis occurs frequently in humans and animals around the world and is caused by Mycoplasmas, which is primarily known as an extracellular pathogen (Atkinson et al., 2008, Chazel et al., 2010, Gambarini et al., 2009, May and Brown, 2009, McVey, 2010, Osman et al., 2009, Sykes, 2010). Mycoplasma gallisepticum (MG), the most important avian pathogenic mycoplasma, causes avian chronic respiratory disease (CRD), especially in chickens and turkeys, which features inflammation of the trachea, air sacs and lungs (Ley, 2003, Stipkovits et al., 2012, Yoder, 1991). The Mycoplasma gallisepticum HS strain (MG-HS) is a virulent strain isolated with a chicken farm in Hubei Province, China (Bi and Ji, 1988, Bi and Xu, 1997). Poultry at different ages can be affected by MG, and young poultry are the most susceptible. After infection, MG usually exists chronically in chicken farms and is very hard to eliminate. Although vaccination and antibiotics can be used to prevent and treat MG infection (Kleven, 2008), and it is impossible to completely clear MG from infected chickens. As a consequence, CRD causes significant economic losses to the poultry industry around the world (Pennycott et al., 2005). In our previous study, we showed that NF-κB is a critical pathway that responds to infection in the chicken lung (Hu et al., 2016, Tian et al., 2016).
Micro-ribonucleic acids (miRNAs), which are 18–26 nucleotides (nt) long, are small non-coding single-stranded RNAs that modulate gene expression at the post-transcriptional level through various mechanisms, including direct cleavage of targeted mRNAs (Bartel, 2004), inhibition of translation (Zhang et al., 2006) or even upregulation of translation (Vasudevan et al., 2007). These activities are mediated through miRNA binding to the 3′ untranslated region (3’UTR) of target mRNAs by base pairing in the miRNA seed region (Valencia-Sanchez et al., 2006, Zamore and Haley, 2005). miRNAs were first discovered in Caenorhabditis elegans (Lee et al., 1993) and were then found to be present in many animals, plants and viruses (Lagos-Quintana et al., 2001, Pfeffer et al., 2004, Reinhart et al., 2005). To date, over 24,000 (in total) and 859 (in chickens) miRNAs have been identified (http://www.mirbase.org/).
miRNAs play important roles in various physiological and pathological processes (Ambros, 2004, Lim et al., 2003). miRNAs are involved in immune processes, the immune response and inflammation modulation (Baltimore et al., 2008, Lindsay, 2008) and certainly participate in various diseases of poultry, including avian influenza (Y et al., 2009), Marek's disease (Li et al., 2014c, Stik et al., 2013), avian leucosis (Li et al., 2014a, Wang et al., 2013a), infectious bursal disease (Y.-s. Wang et al., 2013), and ovarian carcinoma (Lee et al., 2012). All of these studies suggest that miRNAs may play important roles in MG infection in chicken.
Profiling analyses have been employed to detect miRNA changes in tissues infected by pathogenic microorganisms, which could help to reveal molecular pathways that regulate microbial pathogenesis. However, miRNA expression profiles in chickens infected by MG have not been reported so far. Deep sequencing technology, with its high sensitivity, is suited for small RNA discovery (Burnside et al., 2008, Glazov et al., 2008). To identify differentially expressed miRNAs and their target mRNAs, we performed deep sequencing of MG-infected and uninfected lung tissues from SPF chicken embryos in this work. This study contributes to the understanding of the regulatory mechanisms governed by miRNAs and the functional role of miRNAs with MG infection in chicken.
Section snippets
Ethical procedures
Our experimental protocols for chicken-embryo treatment used here were approved by the Institutional Animal Care and Use Committee of Huazhong Agricultural University, P. R. China. The procedures were carried out in accordance with the approved guidelines.
Mycoplasma strains and growth conditions
MG-HS, a virulent strain, was isolated from a chicken farm in Hubei province, China (Bi and Xu, 1997). The strain was deposited and donated by State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong
Overview of the deep Sequencing data
To systematically identify small RNAs and their expression levels in MG-infected and uninfected lung tissues of chicken embryos on day 3 and 10 post-infection (Noiva et al., 2014), four small RNA libraries were constructed from the total RNA of groups I, II, III and IV (see the Methods for the group descriptions) for deep sequencing. A total of 14,305,532, 10,223,342, 13,920,797 and 14,052,956 raw reads were obtained from the group I, group II, group III and group IV libraries, respectively.
Discussion
miRNAs play a critical role in gene regulation. Comprehensive analysis of differentially expressed miRNAs may provide a unique opportunity to enhance our understanding of the regulatory mechanisms in many biological processes, including microbial infection. Small RNA deep sequencing has become an important tool for confirming functional miRNAs. Recent studies have suggested that microbial infections can alter the expression of miRNAs in chickens (Li et al., 2014c, Lian et al., 2015), as well as
Conclusions
This comprehensive analysis provides several lines of new evidence on how host miRNAs might regulate host responses to MG infection in chickens. We identified 45 and 68 differentially expressed miRNAs in phase I and phase II, respectively, and among these, 23 miRNAs were differentially expressed between infected and uninfected tissues at both time points. Eight novel miRNAs were predicted in total. In addition, GO annotation, KEGG pathway, miRNA-GO-network, path-net and gene-net analyses were
Author Contributions
Yabo Zhao and Yue Hou performed the experiments; Yue Hou analysed and interpreted data; Bo Yuan and Kang Zhang performed total RNA isolation and cDNA preparation. Xiuli Peng conceived and designed the study and helped to draft the manuscript. All authors read and approved the final manuscript.
Conflict of interest
All authors declare that we have no conflict of interest.
Acknowledgments
We thank Yangzhang Gong, Yanping Feng, Shijun Li for ideas and support. This study was funded by the National Natural Science Foundation of China (Grant No. 31270216).
References (78)
- et al.
The Kinase Akt1 Controls Macrophage Response to Lipopolysaccharide by Regulating MicroRNAs
Immunity
(2009) MicroRNAs: Genomics, Biogenesis, Mechanism, and Function
Cell
(2004)- et al.
Validation of ATP luminometry for rapid and accurate titration of Mycoplasma hyopneumoniae in Friis medium and a comparison with the color changing units assay
J. Microbiol. Methods
(2010) - et al.
Renal p38 MAP kinase activity in experimental diabetes
Lab. Investig.
(2007) - et al.
microRNA-146 up-regulation predicts the prognosis of non-small cell lung cancer by miRNA in situ hybridization
Exp. Mol. Pathol.
(2014) - et al.
gga-miR-26a targets NEK6 and suppresses Marek's disease lymphoma cell proliferation
Poult. Sci.
(2014) - et al.
Chicken gga-miR-181a targets MYBL1 and shows an inhibitory effect on proliferation of Marek's disease virus-transformed lymphoid cell line
Poult. Sci.
(2015) microRNAs and the immune response
Trends Immunol.
(2008)- et al.
Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2 − ΔΔCT Method
Methods
(2001) - et al.
Wnt/beta-catenin signaling: components, mechanisms, and diseases
Dev. Cell
(2009)
Secreted sialidase activity of canine mycoplasmas
Vet. Microbiol.
Mechano-coupling and regulation of contractility by the vinculin tail domain
Biophys. J.
Frizzled 9 knock-out mice have abnormal B-cell development
Blood
Roles of Toll-like receptors 2 and 6 in the inflammatory response to Mycoplasma gallisepticum infection in DF-1 cells and in chicken embryos
Dev. Comp. Immunol.
Differential expression of microRNAs in avian leukosis virus subgroup J-induced tumors
Vet. Microbiol.
Overexpression of microRNA gga-miR-21 in chicken fibroblasts suppresses replication of infectious bursal disease virus through inhibiting VP1 translation
Antivir. Res.
Computational identification of microRNAs and their targets
Comput. Biol. Chem.
The functions of animal microRNAs
Nature
Epidemiology, clinical manifestations, pathogenesis and laboratory detection of Mycoplasma pneumoniae infections
FEMS Microbiol. Rev.
MicroRNAs: new regulators of immune cell development and function
Nat. Immunol.
Toll-like receptor signaling pathways
Science
Inferences, questions and possibilities in Toll-like receptor signalling
Nature
The isolation and identification of the mycoplasma gallisepticum
Acta Vet. Zootech. Sin.
Study on pathogenicity of HS strain Mycoplasma gallisepticum
Chin. J. Anim. Poult. Infect. Dis.
Deep Sequencing of Chicken microRNAs
BMC Genomics
Mycoplasmoses of ruminants in France: recent data from the national surveillance network
BMC Vet. Res.
MicroRNA-99 Family Members Suppress Homeobox A1 Expression in Epithelial Cells
PLoS One
gga-miR-101-3p Plays a Key Role in Mycoplasma gallisepticum (HS Strain) Infection of Chicken
Int. J. Mol. Sci.
MicroRNA expression during chick embryo development
Dev. Dyn.
Double duty for Rac1 in epidermal wound healing
Sci. STKE
Prognostic significance of miR-126 in various cancers: a meta-analysis
Oncotargets Ther.
Granular Vulvovaginitis Syndrome in Nelore pubertal and post pubertal replacement heifers under tropical conditions: Role of Mycoplasma spp., Ureaplasma diversum and BHV-1
Trop. Anim. Health Prod.
A miR-19 regulon that controls NF-κB signaling
Nucleic Acids Res.
A microRNA catalog of the developing chicken embryo identified by a deep sequencing approach
Genome Res.
Differential regulation of microRNA transcriptome in chicken lines resistant and susceptible to necrotic enteritis disease
Int. J. Remote Sens.
Chicken gga-miR-19a Targets ZMYND11 and Plays an Important Role in Host Defense against Mycoplasma gallisepticum (HS Strain) Infection
Front. Cell. Infect. Microbiol.
Wnt Signaling: Role in Alzheimer Disease and Schizophrenia
J. NeuroImmune Pharmacol.
KEGG for linking genomes to life and the environment
Nucleic Acids Res.
Brassinosteroid regulates stomatal development by GSK3-mediated inhibition of a MAPK pathway
Nature
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2022, Handbook of Epigenetics: The New Molecular and Medical Genetics, Third EditionDF-1 cells prevent MG-HS infection through gga-miR-24-3p/RAP1B mediated decreased proliferation and increased apoptosis
2021, Research in Veterinary ScienceCitation Excerpt :What's more, replication of infectious bursal disease virus (IBDV) is inhibited by miR-130b (Fu et al., 2018) or miR-155(Wang et al., 2020), while it is promoted by miR-9, miR-2127, and miR-142 (Ouyang et al., 2018). Based on the previous studies of our research group, we found that MG could significantly regulate the expression of gga-miR-24-3p in the lung tissue of chicken embryo (Zhao et al., 2017a), which suggested that it may be effective markers for the diagnosis, prognosis and potential therapeutic interventions of the disease. In recent years, numerous studies have shown that miR-24-3p plays an important role in various diseases, especially respiratory disease.
Exosomal miR-181a-5p reduce Mycoplasma gallisepticum (HS strain) infection in chicken by targeting PPM1B and activating the TLR2-mediated MyD88/NF-κB signaling pathway
2021, Molecular ImmunologyCitation Excerpt :The threshold value of statistically significant was p-values of < 0.01 or < 0.05. Our previous miRNAs deep sequencing showed that miR-181a-5p expression increased in the lungs of MG-HS-infected chicken embryos (Zhao et al., 2017). This result was validated again by evaluation of the variation of miR-181a-5p expression after MG-HS infection in both chicken embryos and DF-1 cells.
Down-regulated gga-miR-223 inhibits proliferation and induces apoptosis of MG-infected DF-1 cells by targeting FOXO3
2021, Microbial PathogenesisCitation Excerpt :Hence, it will be crucial to increase our understanding of MG infection mechanisms by studying the potential role of miRNAs involved in MG infection. Based on the previous studies, we found that MG could significantly regulate the expression of multiple miRNAs in the lung tissue of chicken embryo [27], suggesting that these differentially expressed miRNA might be effective markers for the diagnosis and prognosis and potential therapeutic interventions of MG-induced CRD. Previous high-throughput sequencing data showed that gga-miR-223 was signally down-regulated in MG-infected chicken embryo lung [32].
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These authors contributed equally to the study.