Integrated analysis of the miRNA-mRNA regulatory network and role of the miRNA 1971-x in poly-β-hydroxybutyrate (PHB) immunoenhancement in gibel carp (Carassius auratus gibelio).

Poly-β-hydroxybutyrate (PHB), a bacterial-derived homopolymer, has recently emerged as a promising prebiotic with immunostimulatory properties in aquatic animals. However, the immunomodulatory molecular remains unclear. MicroRNAs (miRNAs) play a crucial role in post-transcriptional gene regulation, particularly in immune function. In this study, we investigated the immunoenhancement effects of dietary PHB supplementation in gibel carp (Carassius auratus gibelio) by analyzing miRNA and mRNA expression profiles in the spleen. Results showed that a total of 948 known miRNAs and 471 novel miRNAs in the spleen of PHB-fed and control groups were identified. Among these, 41 differentially expressed (DE) miRNAs were annotated, including 18 miRNAs up-regulated and 23 down-regulated, which targeted 4891 mRNAs. Integrated miRNA-mRNA analysis revealed that PHB enhances immunity via key pathways, including Th1 and Th2 cell differentiation, JAK-STAT signaling, tryptophan metabolism, and B cell receptor signaling pathways. Among DE-miRNAs, the most differential expression of miR-1971-x was chosen to verify the modulation of miRNA on the transcription of immune-related genes, and antioxidant response of leukocytes post PHB treatment. Resultantly, PHB improved the antioxidant capacity of leukocytes from spleen in gibel carp. The miR-1971-x negatively regulated the mRNA expression of genes involved in Th1/Th2 cell differentiation and JAK-STAT signaling pathway. This study provides novel insights into the regulatory role of PHB-induced miRNAs in fish immunity, particularly through miR-1971-x-mediated suppression of Th1/Th2 and JAK-STAT pathways. The findings support the potential of PHB as an eco-friendly immunostimulant in sustainable aquaculture.