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

## TL;DR

This study explores how microRNAs regulate immune priming in oysters, showing that specific miRNAs control haemocyte proliferation and immune response.

## Contribution

The study identifies Cgi-miR-1175-P6/P7-y as a key miRNA regulating immune priming in oysters through post-transcriptional control.

## Key findings

- 67 miRNAs were differentially expressed in both primary and secondary immune stimulations in oysters.
- Cgi-miR-1175-P6/P7-y targets genes involved in cell survival, proliferation, and pattern recognition.
- In vivo experiments confirmed Cgi-miR-1175-P6/P7-y's role in reducing haemocyte proliferation and target gene expression.

## Abstract

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 Crassostrea gigas 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 Vibrio splendidus. Putative immune enhancing miRNAs (Cgi-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 Cgi-miR-1175-P6/P7-y and novel-0095-3p were enriched in cell proliferation-related pathways and metabolic pathways. Target prediction suggests that Cgi-miR-1175-P6/P7-y and novel-m0095-3p may target genes involved in cell survival (CgTEP, CgIAP), cell proliferation (CgCDK6 and CgCDK14) and pattern recognition (CgSCARF2), respectively. Through in vivo injections of Cgi-miR-1175-P6/P7-y mimics, both the rate of EdU+ haemocytes and the mRNA expression levels of its target genes (CgCDK6, CgCDK14 and CgSCARF2) were significantly reduced in mimics-treated group after Vibrio splendidus stimulation, whereas the opposite effects were observed in the Cgi-miR-1175-P6/P7-y inhibitor-treated group. These findings highlight the regulatory role of miRNAs in immune priming and identify Cgi-miR-1175-P6/P7-y as a key post-transcriptional regulator of haemocyte proliferation.