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      MicroRNA profiling of the intestinal tissue of Kazakh sheep after experimental Echinococcus granulosus infection, using a high-throughput approach Translated title: Profilage des microARN du tissu intestinal des moutons Kazakh après infection expérimentale par Echinococcus granulosus, en utilisant une approche à haut débit

      1 , 1 , 1 , 3 , * , 1 , 2 , * , 1 , *

      Parasite

      EDP Sciences

      microRNA, Cystic Echinococcosis, Sheep, MHC haplotype

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          Abstract

          Cystic echinococcosis (CE), caused by infection with the larval stage of the cestode Echinococcus granulosus, is a chronic zoonosis, to which sheep are highly susceptible. Previously, we found that Kazakh sheep with different MHC haplotypes differed in CE infection. Sheep with haplotype MHC MvaIbc- SacIIab- Hin1Iab were resistant to CE infection, while their counterparts without this haplotype were not. MicroRNAs (miRNAs), a class of small non-coding RNAs, are key regulators of gene expression at the post-transcriptional level and play essential roles in fundamental biological processes such as development and metabolism. To identify microRNA controlling resistance to CE in the early stage of infection, microRNA profiling was conducted in the intestinal tissue of sheep with resistant and non-resistant MHC haplotypes after peroral infection with E. granulosus eggs. A total of 351 known and 186 novel miRNAs were detected in the resistant group, against 353 known and 129 novel miRNAs in the non-resistant group. Among these miRNAs, 83 known miRNAs were significantly differentially expressed, including 75 up-regulated and 8 down-regulated miRNAs. Among these known microRNAs, miR-21-3p, miR-542-5p, miR-671, miR-134-5p, miR-26b, and miR-27a showed a significantly higher expression in CE-resistant sheep compared to the CE-non-resistant library, with the FC > 3. Functional analysis showed that they were NF-kB pathway-responsive miRNAs, which are involved in the inflammation process. The results suggest that these microRNAs may play important roles in the response of intestinal tissue to E. granulosus.

          Translated abstract

          L’échinococcose kystique (EK), causée par infection par le stade larvaire du cestode Echinococcus granulosus, est une zoonose chronique, à laquelle les moutons sont particulièrement sensibles. Auparavant, nous avons constaté que les moutons Kazakh avec différents haplotypes de CMH différaient dans l’infection par EK. Les moutons avec l’haplotype MHC MvaIbc-SacIIab-Hin1Iab étaient résistants à l’infection par EK, tandis que leurs homologues sans cet haplotype ne l’étaient pas. Les microARN (miARN), une classe de petits ARN non-codants, sont des régulateurs-clés de l’expression des gènes au niveau post-transcriptionnel et jouent des rôles essentiels dans les processus biologiques fondamentaux tels que le développement et le métabolisme. Pour identifier les microARN contrôlant la résistance à EK dans le stade précoce de l’infection, un profilage des microARN a été réalisé dans le tissu de l’intestin des moutons d’haplotypes MHC résistants et non-résistants, après infection perorale par des œufs d’ E. granulosus. Un total de 351 miARN connus et 186 miARN nouveaux ont été détectés dans le groupe résistant, contre 353 connus et 129 nouveaux dans le groupe non-résistant. Parmi ces miARN, 83 des connus étaient exprimés de manière significativement différente, dont 75 régulés à la hausse et 8 régulés à la baisse. Parmi ces microARN connus, miR-21-3p, miR-542-5p, miR-671, miR-134-5p, miR-26b et miR-27a ont montré une expression significativement plus élevée chez les moutons résistants à EK comparés à la bibliothèque des non-résistants, avec FC > 3. Une analyse fonctionnelle a montré que ce sont des miARN de la voie NF-kB, qui interviennent dans le processus d’inflammation. Les résultats suggèrent que ces microARN peuvent jouer des rôles importants dans la réponse du tissu intestinal à E. granulosus.

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          Most cited references 16

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          MicroRNA-23a/b and microRNA-27a/b suppress Apaf-1 protein and alleviate hypoxia-induced neuronal apoptosis

           Q Chen,  J. Xu,  L. Li (2014)
          Expression of apoptotic protease activating factor-1 (Apaf-1) gradually decreases during brain development, and this decrease is likely responsible for the decreased sensitivity of brain tissue to apoptosis. However, the mechanism by which Apaf-1 expression is decreased remains elusive. In the present study, we found that four microRNAs (miR-23a/b and miR-27a/b) of miR-23a-27a-24 and miR-23b-27b-24 clusters play key roles in modulating the expression of Apaf-1. First, we found that miR-23a/b and miR-27a/b suppressed the expression of Apaf-1 in vitro. Interestingly, the expression of the miR-23-27-24 clusters in the mouse cortex gradually increased in a manner that was inversely correlated with the pattern of Apaf-1 expression. Second, hypoxic injuries during fetal distress caused reduced expression of the miR-23b and miR-27b that was inversely correlated with an elevation of Apaf-1 expression during neuronal apoptosis. Third, we made neuronal-specific transgenic mice and found that overexpressing the miR-23b and miR-27b in mouse neurons inhibited the neuronal apoptosis induced by intrauterine hypoxia. In conclusion, our results demonstrate, in central neural system, that miR-23a/b and miR-27a/b are endogenous inhibitory factors of Apaf-1 expression and regulate the sensitivity of neurons to apoptosis. Our findings may also have implications for the potential target role of microRNAs in the treatment of neuronal apoptosis-related diseases.
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            NF-kappaB p65-Dependent Transactivation of miRNA Genes following Cryptosporidium parvum Infection Stimulates Epithelial Cell Immune Responses

            Introduction The protozoan parasite, Cryptosporidium parvum, is a causative agent of human gastrointestinal disease worldwide [1]. C. parvum infects the gastrointestinal epithelium to produce a self-limiting diarrhea in immunocompetent individuals but is potentially life-threatening in immunocompromised persons, especially those with the acquired immunodeficiency syndrome (AIDS) [1],[2]. Transmission occurs via the fecal-oral route. Humans are infected by ingesting C. parvum oocysts; oocysts then excyst in the gastrointestinal tract releasing infective sporozoites. C. parvum sporozoites can also travel up the biliary tract to infect the epithelial cells lining the biliary tract (i.e. cholangiocytes) [1],[3]. Mediated by specific ligands on the sporozoite surface and receptors on the host cells, the sporozoite attaches to the apical membrane of epithelial cells and forms a parasitophorous vacuole in which the organism remains intracellular but extracytoplasmic [3]. The sporozoite then matures and undergoes further development of its life cycle. With this unique extracytoplasmic niche within epithelial cells preventing a direct infection of other cell types, C. parvum is classified as a “minimally invasive” mucosal pathogen [1]. Because of the “minimally invasive” nature of C. parvum infection, innate immune responses by epithelial cells are critical to the host's defense against infection. Toll-like receptor (TLR) - and nuclear factor-kappaB (NF-κB) -mediated signaling pathways are important components in epithelial innate immunity to C. parvum infection [4],[5]. TLRs are transmembrane proteins with highly conserved structural domains [6]. Upon engagement of the TLRs by specific ligands, various adaptor molecules including myeloid differentiation factor 88 (MyD88) are selectively recruited to the receptors forming a complex referred to as the “signalosome” [6],[7]. The signalosome then triggers a series of downstream events including activation of the NF-κB [6]–[8]. NF-κB subunits bind to the κB sites within the promoters/enhancers of target genes resulting in the transcriptional regulation of multiple genes important to epithelial anti-C. parvum defense [4],[5]. MicroRNAs (miRNAs), a newly identified class of endogenous small regulatory RNAs of ∼24 nucleotides, are emerging as key mediators of many biological processes and impact gene expression at the posttranscriptional level [9],[10]. Similar to other RNA molecules, most of miRNAs are initially transcribed as primary transcripts (termed pri-miRNAs) by Poly II and processed by the RNase III Drosha (in the nucleus) and a second RNase III Dicer (in the cytoplasm) to generate mature miRNA molecules [11]–[13]. However, molecular mechanisms underlying miRNA gene transcriptional regulation are largely unclear [14]. Recent studies on expression of miRNA genes have revealed potential transcriptional regulation by transcription factors, such as NF-κB and C/EBPα [15],[16]. While much of our understanding of the cellular processes modulated by miRNAs has come from studies on development and tumorigenesis, the role of miRNAs in immune responses is now being gradually uncovered [17]–[19]. The importance of miRNAs in cell-mediated immunity is highlighted by Dicer conditional knockout mice. Specific deletion of dcr-1 in the T cell lineage resulted in impaired T cell development and aberrant T helper cell differentiation and cytokine production [20]. In addition, miRNA expression is impacted by cytokines in some model systems. Both interferon (IFN) -α and IFN-β modulate expression of several miRNAs required for their anti-viral responses following infection with hepatitis C virus [21]. The TLR4 ligand, lipopolysaccharide (LPS), impacts expression of miR-132, miR-146, and miR-155 in human THP-1 monocytes [15],[22]. Further characterization of miR-146 revealed that this miRNA may function as a negative regulator of tumor necrosis factor receptor-associated factor 6 and interleukin-1 receptor associated kinase 1 [15]. Recent studies also implicate specific miRNAs in controlling various epithelial cell processes such as regulation of cellular differentiation, determination of epithelial cell fate (cell death and proliferation), initiation and regulation of anti-microbial immunity, fine-tuning of inflammatory responses, and activation of specific intracellular signaling pathways [17]–[19],[23]. Using a non-malignant human cholangiocyte cell line (H69) that expresses multiple TLRs including TLR4 [5], we previously demonstrated that infection of human cholangiocytes by C. parvum in vitro mimics parasitial apical invasion and TLR4/NF-κB-dependent epithelial responses in vivo [3]. Moreover, let-7 regulates TLR4 expression via translational suppression in human cholangiocytes and is involved in epithelial defenses against C. parvum [24]. Members of the miR-98/let-7 family also regulate expression of cytokine-inducible SH2-containing protein (CIS) in cholangiocytes following C. parvum infection [25]. Together, these findings demonstrate that miRNAs levels in epithelial cells are altered by C. parvum infection and may regulate epithelial anti-C. parvum immune responses. In this study, we performed an array analysis of miRNA expression in H69 cells following C. parvum infection and LPS stimulation. The analysis revealed significant alterations in miRNA expression in cholangiocytes following C. parvum infection or treatment with LPS. Of those miRNAs upregulated by C. parvum infection, we identified potential NF-κB binding sites in the promoter elements of several miRNA genes. Inhibiting activation of NF-κB p65 blocked C. parvum-induced upregulation of a panel of miRNA genes. Promoter binding and transactivation of the NF-κB p65 subunit of each selected miRNA gene was confirmed by chromatin immunoprecipitation assay and promoter luciferase reporter analysis. Furthermore, functional inhibition of the NF-κB p65-binding miRNAs increased C. parvum burden in cholangiocytes in vitro. These data demonstrate that a panel of miRNAs is regulated through promoter binding of the NF-κB p65 subunit in human cholangiocytes and these miRNAs are involved in epithelial defense in response to C. parvum infection, suggesting a role of miRNAs in regulation of epithelial anti-microbial defense. Results C. parvum infection induces alterations in miRNA expression in cholangiocytes in vitro To globally assess miRNA expression in epithelial cells following C. parvum infection, we performed a microarray analysis of mature miRNA expression in H69 cells [26]. The miRCURY™ LNA human microRNAs assays (version 8.1; Exiqon; Vedbaek, Denmark) covers a total of up to 600 known human mature miRNAs and were used as previously described [27]. The quality of the RNA was verified using an Agilent 2100 Bioanalyzer (Figure S1). A total of 383 mature miRNAs were detected in the uninfected H69 cells. Of the miRNAs expressed, miR-23b, miR-30b, miR-30c, and miR-125b expression were significantly increased in H69 cells after exposure to live C. parvum infection for 12 h (p< = 0.05; Figure 1A and Table S1). Five additional miRNAs (miR-15b, miR-16, miR-27b, miR-24, and miR-21) showed a tendency to increase (0.05
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              MicroRNA-542-5p as a novel tumor suppressor in neuroblastoma.

              Several studies have implicated the dysregulation of microRNAs in neuroblastoma pathogenesis, an often fatal paediatric cancer arising from precursor cells of the sympathetic nervous system. Our group and others have demonstrated that lower expression of miR-542-5p is highly associated with poor patient survival, indicating a potential tumor suppressive function. Here, we demonstrate that ectopic over-expression of this miRNA decreases the invasive potential of neuroblastoma cell lines in vitro, along with primary tumor growth and metastases in an orthotopic mouse xenograft model, providing the first functional evidence for the involvement of miR-542-5p as a tumor suppressor in any type of cancer. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.
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                Author and article information

                Journal
                Parasite
                Parasite
                parasite
                Parasite
                EDP Sciences
                1252-607X
                1776-1042
                2016
                27 May 2016
                : 23
                : ( publisher-idID: parasite/2016/01 )
                Affiliations
                [1 ] College of Animal Science and Technology, Shihezi University Road Beisi Shihezi 832003 Xinjiang PR China
                [2 ] Institute of Animal Husbandry and Veterinary Medicine, Anhui Academy of Agriculture Sciences Road Nongkenan Hefei 230031 Anhui PR China
                [3 ] Center for Stem Cell and Translational Medicine, School of Life Sciences, Anhui University Road Jiulong Hefei 230000 Anhui PR China
                Author notes
                [§]

                1 and 2 contributed equally.

                Article
                parasite160024 10.1051/parasite/2016023
                10.1051/parasite/2016023
                4884269
                27235195
                © S. Jiang et al., published by EDP Sciences, 2016

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                Page count
                Figures: 1, Tables: 3, Equations: 0, References: 29, Pages: 8
                Categories
                Research Article

                microrna, cystic echinococcosis, sheep, mhc haplotype

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