MicroRNAs (miRNAs) are closely related to the occurrence, development, and immune response of diseases. BCL2-associated athanogene 2 (BAG2) is a member of the BAG family that functions in diverse cellular processes, including cell death, differentiation, and cell division. In this study, we cloned the cDNA full-length of sea cucumber (Apostichopus japonicus) BAG2 (AjBAG2) and confirmed it is an anti-apoptotic protein in vitro and in vivo during Vibrio splendidus infection. Moreover, we identified a perfect complementarity between miR-375 and the 3'-untranslated region (UTR) sequence of AjBAG2. The miR-375 expression decreased the luciferase activity dose-dependently when co-transfected with the AjBAG2 3'-UTR-luciferase reporter containing the miR-375 target site in epithelioma papulosum cyprini (EPC) cells. This inhibition was partially recovered by a miR-375 specific inhibitor. The mRNA and protein levels of AjBAG2 were opposite to that of coelomocytes in challenged sea cucumber when treated with miR-375 mimics or inhibitors. Additionally, miR-375 expression induced coelomocytes apoptosis and blocked the anti-apoptotic activity of AjBAG2. Our data demonstrated that AjBAG2 is an anti-apoptotic protein during V. splendidus infection and this function can be inhibited by miR-375 in sea cucumbers.

RhoA (Ras homolog A) protein is a representative member of the Rho GTPase family and is involved in various cellular processes. The function of RhoA in sea cucumbers is unclear. In this study, we hypothesized that RhoA may regulate the innate immune response of Apostichopus japonicus. Our data showed that 1) the complete sequence of RhoA from A. japonicus (named AjRhoA) was 968 bp, with a high level sequence conservation across the echinoderms and other phyla; 2) tissue expression analysis showed that AjRhoA transcripts and protein exhibited higher abundance in coelomocytes, whereas the relative expression of miR-2012-5p was lower in coelomocytes; 3) interactive binding sites and a negative regulatory targeting relationship between AjRhoA and miR-2012-5p were confirmed through a dual-luciferase reporter assay and functional validation in vivo; 4) the relative expression levels of AjRhoA transcripts and protein were upregulated in coelomocytes 4- and 72-hour post infection (hpi) with Vibrio splendidus, whereas miR-2012-5p was expressed in the opposite pattern; 5) both AjRhoA silencing and miR-2012-5p overexpression suppressed the phagocytic capacity of A. japonicus compared with the control at 4 and 72 hpi. Our observations suggest that AjRhoA can regulate the pathogen-induced immune response of A. japonicus through the "AjRhoA-miR-2012-5p" module during the early infection, while miR-2012-5p plays a direct immunomodulatory role as the infection progresses.