Identification of a novel RhoA gene in the sea cucumber Apostichopus japonicus and its immune regulatory function via interacting with miR-2012-5p.

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.