The involvement of miRNAs in the enhanced immune response of oysters via haemocyte-mediated immune priming

Immune priming enhances innate immunity, leading to a sustained and augmented response upon secondary challenge. The emerging evidence has highlighted the crucial role of endogenous microRNAs in trained immunity of vertebrates. However, the regulatory role of miRNAs in immune priming of invertebrates remains largely unknown. In the present study, the miRNA expression profile in the haemocyte-mediated immune priming of oysters <i>Crassostrea gigas</i> was examined. There were 115 up- and 212 down-regulated miRNAs screened after primary stimulation, and 107 up- and 103 down-regulated miRNAs identified after secondary stimulation. Among these, 67 miRNAs were differentially expressed in both the primary and secondary stimulations of <i>Vibrio</i> sp<i>lendidus</i>. Putative immune enhancing miRNAs (<i>Cgi</i>-miR-1175-P6/P7-y and novel-0095-3p) showed lower expression upon secondary stimulation compared to the primary response. KEGG analysis indicated that target genes of <i>Cgi</i>-miR-1175-P6/P7-y and novel-0095-3p were enriched in cell proliferation-related pathways and metabolic pathways. Target prediction suggests that <i>Cgi</i>-miR-1175-P6/P7-y and novel-m0095-3p may target genes involved in cell survival (<i>Cg</i>TEP, <i>Cg</i>IAP), cell proliferation (<i>Cg</i>CDK6 and <i>Cg</i>CDK14) and pattern recognition (<i>Cg</i>SCARF2), respectively. Through <i>in vivo</i> injections of <i>Cgi</i>-miR-1175-P6/P7-y mimics, both the rate of EdU⁺ haemocytes and the mRNA expression levels of its target genes (<i>Cg</i>CDK6, <i>Cg</i>CDK14 and <i>Cg</i>SCARF2) were significantly reduced in mimics-treated group after <i>Vibrio</i> sp<i>lendidus</i> stimulation, whereas the opposite effects were observed in the <i>Cgi</i>-miR-1175-P6/P7-y inhibitor-treated group. These findings highlight the regulatory role of miRNAs in immune priming and identify <i>Cgi</i>-miR-1175-P6/P7-y as a key post-transcriptional regulator of haemocyte proliferation.