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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
.
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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).
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