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.

环状 RNA (circRNA) 是一种广泛多样的共价闭合环状内源性 RNA，在哺乳动物的免疫调节中发挥着至关重要的作用。然而，circRNA 在无脊椎动物中的功能和机制在很大程度上尚不清楚。在我们之前的工作中，通过 RNA-seq从皮肤溃疡综合征 (SUS) 病海参 Apostichopus japonicus 中鉴定出包括 circRNA432 (circ432) 在内的 261 个差异表达的 circRNA 。为了更好地解决海参 circRNA 的功能作用，本研究首次发现 circ432 被灿烂弧菌攻击和 LPS 暴露显着诱导。敲除 circ432 可能会抑制V. splendidus-诱导的体腔细胞吞噬作用。此外，通过 Argonaute 2-RNA 免疫沉淀 (AGO2-RIP) 测定、荧光素酶报告基因测定和 RNA 荧光原位杂交 (FISH)，circ432 被验证为 miR-2008 的海绵，miR-2008 是 SUS 病海参中差异表达的 miRNA ） 体外。吞噬和细胞运动蛋白 1 (AjELMO1) 被进一步证明是 miR-2008 的靶标，沉默 AjELMO1 会抑制V. splendidus诱导的体腔细胞吞噬作用，通过补充 miR-2008 模拟物可以进一步抑制这种现象，表明 circ432 可能通过 miR-2008-AjELMO1 轴调节体腔细胞吞噬作用。我们进一步证实，通过 circ432 沉默抑制的体腔细胞吞噬作用与 AjELMO1 丰度降低一致，并且可以通过 miR-2008 抑制剂转染恢复。我们所有的结果都证明 circ432 通过海绵 miR-2008 参与调节病原体诱导的体腔细胞吞噬作用，并促进 AjELMO1 的丰度。这些研究结果将丰富棘皮动物吞噬作用的调控机制，为海参SUS病害防治提供理论数据。