Click
here to close Hello! We notice that
you are using Internet Explorer, which is not supported by Echinobase
and may cause the site to display incorrectly. We suggest using a
current version of Chrome,
FireFox,
or Safari.
miRNA-133 augments coelomocyte phagocytosis in bacteria-challenged Apostichopus japonicus via targeting the TLR component of IRAK-1 in vitro and in vivo.
Lu M
,
Zhang PJ
,
Li CH
,
Lv ZM
,
Zhang WW
,
Jin CH
.
???displayArticle.abstract???
In this study, we explored the potential roles of miRNA-133 in regulating TLR pathways in the sea cucumber Apostichopus japonicus. Target screening of RNA-Seq data successfully identified interleukin-1 receptor-associated kinase (AjIRAK-1) as a putative target of miR-133. This result was further validated by negative expression profiles in Vibrio splendidus-challenged coelomocytes and lipopolysaccharide (LPS)-exposed cell cultures. HEK-293T cells transfected with a dual-luciferase reporter fused to the 3''UTR of wild-type or mutant AjIRAK-1 exhibited a 52.9% reduction in luciferase activity (p < 0.01) compared to controls. Co-infection with a miR-133 mimics or a specific siRNA targeting AjIRAK-1 significantly repressed the mRNA and protein expression levels of AjIRAK-1 and its downstream molecules, such as AjTRAF6 and Ajp105, in primary coelomocytes. In contrast, a miR-133 inhibitor significantly increased the expression of these TLR pathway members. The injection of miR-133 agomir or AjIRAK-1 siRNA into sea cucumbers not only decreased the expression of AjIRAK-1 and its downstream molecules but also significantly increased V. splendidus coelomocyte phagocytosis. All of the present data provide direct evidence that miR-133 is involved in TLR cascade modulation through AjIRAK-1 targeting to promote V. splendidus coelomocyte phagocytosis in these non-model invertebrates.
Figure 1. The complete cDNA sequence of interleukin-1 receptor-associated kinase 1 from Apostichopus japonicus and its predicted amino acid sequence.A typical death domain (DD) and a kinase domain are shadowed and underlined, respectively. Three highly conserved motifs are shown boxed. The ATP-binding site is italicized and bolded. The kinase-action site is bolded. The two putative biding sites of miR-133 are inlicited and bolded.
Figure 2. The phylogenetic tree constructed based on the IRAK amino acid sequence using the neighbor-joining algorithm.The GenBank accession numbers used are as follows:Ornithorhynchus anatinus IRAK-1 XP_007667869; Amphimedon queenslandica IRAK-4 XP_003388757; Lepisosteus oculatus IRAK-4 XP_006642971; Aplysia californica IRAK-1 XP_005095880; Caenorhabditis elegans IRAK-1 NP_001255742; Aplysia californica IRAK-1 XP_005095879; Bubalus bubalis IRAK-1 XP_006043774; Salmo salar IRAK-4 NP_001135238; Meleagris gallopavo IRAK-4 XP_003202068; Columba livia IRAK-4 XP_005499983; Epinephelus coioides IRAK-4 AGQ48127; Litopenaeus vannamei IRAK-1 AGU41814; Oncorhynchus mykiss IRAK-4 CBI63176; Melopsittacus undulates IRAK-4 XP_005148151; Alligator sinensis IRAK-4 XP_006015992; Loxodonta Africana IRAK-1 XP_003421767; Danio rerio IRAK-1 XP_005166760; Condylura cristata IRAK-1 XP_004695245; Ficedula albicollis IRAK-4 XP_005039421; Echinops telfairi IRAK-1 XP_004717621; Alligator mississippiensis IRAK-4 XP_006272300; Bos mutus IRAK-1 ELR46602; Coregonus maraena IRAK-4 CBI63179; Pan paniscus IRAK-4 XP_003825792; Homo sapiens IRAK-4 AAM15772.
Figure 3. The time-course expression patterns of miR-133 and AjIRAK-1 in V. splendidus-challenged A. japonicus (A,B)—and LPS-exposed primary cultured cells (C,D) as measured by qRT-PCR. Data are expressed as the mean ± SD (n = 3).
Figure 4. A schematic of the luciferase reporter assay used to validate the interaction between miR-133 and the 3′UTR of AjIRAK-1.The bold font indicates the “seed†regions. The AjIRAK-1 3′UTRs of the wild type and full mutants are shown in A and B. A: Two predicted miR-133 binding sites in the AjIRAK-1 3′UTR. B: Mutant sequences of the AjIRAK-1 3′UTR seed sequence. WT: Wild-type; MT: Mutant type. C:The relative expression levels of miR-133 after miR-133 mimics and plasmid coinfection. NCM: Negative control of miR-133 mimics; 133M: miR-133 mimics; 133M + WT: miR-133 mimics and wild-type plasmid coinfection; 133M + MT: miR-133 mimics and mutant-type plasmid coinfection; D: Normalized luciferase activity of a reporter containing the WT or MT reporter constructs of AjIRAK-1 in HEK293T cells co-transfected with negative control of miR-133 mimics (NCM) or miR-133 mimics (133M). The data are represented as the mean ± SD (n = 6).
Figure 5. The expression profiles of miR-133, AjIRAK-1 and the downstream signaling molecules after transfection with miR-133 inhibitors(B,G–J) or mimics (A,C–F) in primary cultured coelomocytes. Three biological replicates were performed in the experiment and the obtained data are expressed as the mean ± SD (n = 3). NCM: miR-133 mimics control; NCI: miR-133 inhibitor control; 133M: miR-133 mimics; 133I: miR-133 inhibitor.
Figure 6. Western blot analysis of AjIRAK-1 protein expression after miR-133 abberrant expression and AjIRAK-1 interference in primary coelomocytes.NCM: Negative control of miR-133 mimics; 133M: miR-133 mimics; NCI: Negative control for the miR-133 inhibitor; 133I: miR-133 inhibitor; siNC: Negative control for the AjIRAK-1 siRNA; siAjIRAK-1: AjIRAK-1 interference.
Figure 7. The mRNA expression levels of AjIRAK-1 and its downstream molecules after AjIRAK-1 interference using siRNA.Three biological replicates were performed in the experiment and the obtained data are expressed as the mean ± SD (n = 3). siAjIRAK-1: AjIRAK-1 siRNA transfection; siNC: control siRNA transfection.
Figure 8. The mRNA expression levels of AjIRAK-1 and its downstream molecules after miR-133 agomir or AjIRAK-1 interference in vivo.Three biological replicates were performed in the experiment and the obtained data are expressed as the mean ± SD (n = 3). 133A: miR-133 agomir; NCA: Negative control of miR-133 agomir;siAjIRAK-1: AjIRAK-1 siRNA transfection; siNC: control siRNA transfection.
Figure 9. Coelomocyte phagocytosis activity after miR-133 agomir or AjIRAK-1 siRNA injection in vivo by the CFU assay.Three biological replicates were performed in the experiment and the obtained data are expressed as the mean ± SD (n = 3).
Abdellatif,
The role of microRNA-133 in cardiac hypertrophy uncovered.
2010, Pubmed
Abdellatif,
The role of microRNA-133 in cardiac hypertrophy uncovered.
2010,
Pubmed
Bartel,
MicroRNAs: genomics, biogenesis, mechanism, and function.
2004,
Pubmed
Baud,
Is NF-kappaB a good target for cancer therapy? Hopes and pitfalls.
2009,
Pubmed
Blander,
Regulation of phagosome maturation by signals from toll-like receptors.
2004,
Pubmed
Boldin,
MicroRNAs, new effectors and regulators of NF-κB.
2012,
Pubmed
Busillo,
Regulation of CXCR4 signaling.
2007,
Pubmed
Chen,
HCV-induced miR-21 contributes to evasion of host immune system by targeting MyD88 and IRAK1.
2013,
Pubmed
Chu,
MicroRNA-630 is a prognostic marker for patients with colorectal cancer.
2014,
Pubmed
Duan,
miR-133b, a muscle-specific microRNA, is a novel prognostic marker that participates in the progression of human colorectal cancer via regulation of CXCR4 expression.
2013,
Pubmed
Furuta,
DNA demethylation-dependent enhancement of toll-like receptor-2 gene expression in cystic fibrosis epithelial cells involves SP1-activated transcription.
2008,
Pubmed
Hajishengallis,
Intracellular signaling and cytokine induction upon interactions of Porphyromonas gingivalis fimbriae with pattern-recognition receptors.
2004,
Pubmed
He,
MicroRNAs: new regulators of Toll-like receptor signalling pathways.
2014,
Pubmed
Hou,
MicroRNA-146a feedback inhibits RIG-I-dependent Type I IFN production in macrophages by targeting TRAF6, IRAK1, and IRAK2.
2009,
Pubmed
John,
Human MicroRNA targets.
2004,
Pubmed
Kong,
MyD88-independent activation of a novel actin-Cdc42/Rac pathway is required for Toll-like receptor-stimulated phagocytosis.
2008,
Pubmed
Koutsoulidou,
Expression of miR-1, miR-133a, miR-133b and miR-206 increases during development of human skeletal muscle.
2011,
Pubmed
Lee,
Characterization of a novel molluscan MyD88 family protein from manila clam, Ruditapes philippinarum.
2011,
Pubmed
Li,
Bacterial lipoprotein-induced self-tolerance and cross-tolerance to LPS are associated with reduced IRAK-1 expression and MyD88-IRAK complex formation.
2006,
Pubmed
Li,
A manganese superoxide dismutase in blood clam Tegillarca granosa: molecular cloning, tissue distribution and expression analysis.
2011,
Pubmed
Li,
Characterization of skin ulceration syndrome associated microRNAs in sea cucumber Apostichopus japonicus by deep sequencing.
2012,
Pubmed
,
Echinobase
Liu,
Identification of the pathogens associated with skin ulceration and peristome tumescence in cultured sea cucumbers Apostichopus japonicus (Selenka).
2010,
Pubmed
,
Echinobase
Lu,
Characterization of two negative regulators of the Toll-like receptor pathway in Apostichopus japonicus: inhibitor of NF-κB and Toll-interacting protein.
2013,
Pubmed
,
Echinobase
Lu,
Two adaptor molecules of MyD88 and TRAF6 in Apostichopus japonicus Toll signaling cascade: molecular cloning and expression analysis.
2013,
Pubmed
,
Echinobase
Ma,
MicroRNAs in NF-kappaB signaling.
2011,
Pubmed
Medvedev,
Dysregulation of LPS-induced Toll-like receptor 4-MyD88 complex formation and IL-1 receptor-associated kinase 1 activation in endotoxin-tolerant cells.
2002,
Pubmed
Moretti,
Insights into phagocytosis-coupled activation of pattern recognition receptors and inflammasomes.
2014,
Pubmed
Morley-Smith,
Circulating microRNAs for predicting and monitoring response to mechanical circulatory support from a left ventricular assist device.
2014,
Pubmed
Muñoz-Culla,
Blood miRNA expression pattern is a possible risk marker for natalizumab-associated progressive multifocal leukoencephalopathy in multiple sclerosis patients.
2014,
Pubmed
Mutallip,
Glutathione S-transferase P1 (GSTP1) suppresses cell apoptosis and its regulation by miR-133α in head and neck squamous cell carcinoma (HNSCC).
2011,
Pubmed
Neal,
Enterocyte TLR4 mediates phagocytosis and translocation of bacteria across the intestinal barrier.
2006,
Pubmed
Nohata,
microRNA-1/133a and microRNA-206/133b clusters: dysregulation and functional roles in human cancers.
2012,
Pubmed
Peng,
Clinical impact of circulating miR-133, miR-1291 and miR-663b in plasma of patients with acute myocardial infarction.
2014,
Pubmed
Qazi,
Microbial heat shock protein 70 stimulatory properties have different TLR requirements.
2007,
Pubmed
Qiu,
Identification and characterization of a myeloid differentiation factor 88 (MyD88) cDNA from Zhikong scallop Chlamys farreri.
2007,
Pubmed
Qiu,
MiR-145, miR-133a and miR-133b inhibit proliferation, migration, invasion and cell cycle progression via targeting transcription factor Sp1 in gastric cancer.
2014,
Pubmed
Rauta,
Toll-like receptors (TLRs) in aquatic animals: signaling pathways, expressions and immune responses.
2014,
Pubmed
Sanjuan,
Toll-like receptor signaling in the lysosomal pathways.
2009,
Pubmed
Sun,
Identification and expression analysis of two Toll-like receptor genes from sea cucumber (Apostichopus japonicus).
2013,
Pubmed
,
Echinobase
Taganov,
NF-kappaB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses.
2006,
Pubmed
Takeuchi,
Pattern recognition receptors and inflammation.
2010,
Pubmed
Tao,
microRNA-133 inhibits cell proliferation, migration and invasion in prostate cancer cells by targeting the epidermal growth factor receptor.
2012,
Pubmed
Tili,
Expression and function of micro-RNAs in immune cells during normal or disease state.
2008,
Pubmed
Uchida,
MiR-133a induces apoptosis through direct regulation of GSTP1 in bladder cancer cell lines.
2013,
Pubmed
Virtue,
MicroRNAs and toll-like receptor/interleukin-1 receptor signaling.
2012,
Pubmed
Wang,
Aj-rel and Aj-p105, two evolutionary conserved NF-κB homologues in sea cucumber (Apostichopus japonicus) and their involvement in LPS induced immunity.
2013,
Pubmed
,
Echinobase
Wang,
Litopenaeus vannamei Toll-interacting protein (LvTollip) is a potential negative regulator of the shrimp Toll pathway involved in the regulation of the shrimp antimicrobial peptide gene penaeidin-4 (PEN4).
2013,
Pubmed
Wen,
Shrimp MyD88 responsive to bacteria and white spot syndrome virus.
2013,
Pubmed
Wu,
Airway epithelial cell tolerance to Pseudomonas aeruginosa.
2005,
Pubmed
Wu,
Human glutathione S-transferase P1-1 interacts with TRAF2 and regulates TRAF2-ASK1 signals.
2006,
Pubmed
Xu,
The muscle-specific microRNAs miR-1 and miR-133 produce opposing effects on apoptosis by targeting HSP60, HSP70 and caspase-9 in cardiomyocytes.
2007,
Pubmed
Yin,
Regulation of zebrafish heart regeneration by miR-133.
2012,
Pubmed
Zhang,
De novo assembly of the sea cucumber Apostichopus japonicus hemocytes transcriptome to identify miRNA targets associated with skin ulceration syndrome.
2013,
Pubmed
,
Echinobase
