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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.

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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