Many organisms have adopted a conserved Toll-like receptor(TLR)signaling pathway in their innate immune system to defend against the invasion of pathogenic microorganisms.However,the underlying regulatory mechanism is not well understood,especially in invertebrates.In this study,we explored the potential roles of miRNA-133 in regulating TLR pathways in the sea cucumber Apostichopus japonicus.A target screening of RNA-seq data successfully identified interleukin-1 receptor-associated kinases(AjIRAK-1)as one of putative targets of miR-133.This result was further validated by the negative expression profiles in Vibrio splendidus-challenged coelomocytes and LPS-exposed cell culture.Using a dual-luciferase reporter fused to the 3UTR of wild type or mutant AjIRAK-1 and the mutant,we found a 52.9%reduction in the luciferase activity in transfected HEK293 cells(p<0.01)when compared to controls.Co-infection with miR-133 mimics or specific siRNA of AjIRAK-1 that significantly repressed the expression of AjIRAK-1 and its down-stream molecules,such as AjTRAF6,AjIκB and Ajp105,in primary coelomocytes.In contrast,a miR-133 inhibitor significantly increased the expression of these TLR-pathway members.All of the data provide firsthand evidence that miR-133 modulates TLR-based immune response through the targeting of AjIRAK-1 in this non-model invertebrates.