title: miRNA-133 augments coelomocyte phagocytosis in bacteria-challenged Apostichopus japonicus via targeting the TLR component of IRAK-1 in vitro and in vivo.
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.
authors.affiliation: School of Marine Sciences, Ningbo University, Ningbo, Zhejiang Province 315211, P.R China.
authors.forename: Meng
authors.initials: M
authors.lastname: Lu
authors.affiliation: School of Marine Sciences, Ningbo University, Ningbo, Zhejiang Province 315211, P.R China.
authors.forename: Peng-Juan
authors.initials: PJ
authors.lastname: Zhang
authors.affiliation: School of Marine Sciences, Ningbo University, Ningbo, Zhejiang Province 315211, P.R China.
authors.forename: Cheng-Hua
authors.initials: CH
authors.lastname: Li
authors.affiliation: School of Marine Sciences, Ningbo University, Ningbo, Zhejiang Province 315211, P.R China.
authors.forename: Zhi-Meng
authors.initials: ZM
authors.lastname: Lv
authors.affiliation: School of Marine Sciences, Ningbo University, Ningbo, Zhejiang Province 315211, P.R China.
authors.forename: Wei-Wei
authors.initials: WW
authors.lastname: Zhang
authors.affiliation: School of Marine Sciences, Ningbo University, Ningbo, Zhejiang Province 315211, P.R China.
authors.forename: Chun-Hua
authors.initials: CH
authors.lastname: Jin
mesh_terms: D020413:3' Untranslated Regions; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D017930:Genes, Reporter; D057809:HEK293 Cells; D006801:Humans; D053592:Interleukin-1 Receptor-Associated Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000633:toxicity; D035683:MicroRNAs / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000378:metabolism; D010587:Phagocytosis / Q000187:drug effects; D010802:Phylogeny; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012616:Sea Cucumbers / Q000166:cytology / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D051193:Toll-Like Receptors / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D014733:Vibrio / Q000472:pathogenicity*
chemical_list: D020413:3' Untranslated Regions; D008070:Lipopolysaccharides; D035683:MicroRNAs; D016376:Oligonucleotides, Antisense; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D051193:Toll-Like Receptors; D004247:DNA; D053592:Interleukin-1 Receptor-Associated Kinases