circRNA432 enhances the coelomocyte phagocytosis via regulating the miR-2008-ELMO1 axis in Vibrio splendidus-challenged Apostichopus japonicus

Circular RNAs (circRNAs) are a kind of extensive and diverse covalently closed circular endogenous RNA, which exert crucial functions in immune regulation in mammals. However, the functions and mechanisms of circRNAs in invertebrates are largely unclarified. In our previous work, 261 differentially expressed circRNAs including circRNA432 (circ432) were identified from skin ulcer syndrome (SUS) diseased sea cucumber Apostichopus japonicus by RNA-seq. To better address the functional role of sea cucumber circRNAs, circ432 was first found to be significantly induced by Vibrio splendidus challenge and LPS exposure in this study. Knock-down circ432 could depress the V. splendidus-induced coelomocytes phagocytosis. Moreover, circ432 is validated to serve as the sponge of miR-2008, a differential expressed miRNA in SUS-diseased sea cucumbers, by Argonaute 2-RNA immunoprecipitation (AGO2-RIP) assay, luciferase reporter assay and RNA fluorescence in situ hybridization (FISH) in vitro. Engulfment and cell motility protein 1 (AjELMO1) is further demonstrated to be the target of miR-2008, and silencing AjELMO1 inhibits the V. splendidus-induced coelomocytes phagocytosis, and this phenomenon could be further suppressed by supplementing with miR-2008 mimics, suggesting that circ432 might regulate coelomocytes phagocytosis via miR-2008-AjELMO1 axis. We further confirm that the depressed coelomocytes' phagocytosis by circ432 silencing is consistent with the decreased abundance of AjELMO1, and could be recovered by miR-2008 inhibitors transfection. All our results provide the evidence that circ432 is involved in regulating pathogen-induced coelomocyte phagocytosis via sponge miR-2008 and promotes the abundance of AjELMO1. These findings will enrich the regulatory mechanism of phagocytosis in echinoderm and provide theoretical data for SUS disease prevention and control in sea cucumbers. The study of the role of circRNA432 in a sea cucumber expands our knowledge about the relationship between circRNA and miRNA in marine invertebrates and highlights the functional links between the non-coding RNA and the pathogen-induced phagocytosis.