Dietary Vitamin D₃ Mediates MiR-194a In The JAK-STAT Immune Pathway Of Yellow Catfish Pelteobagrus Fulvidraco

Micro RNA（mi RNA）is a non-coding single-stranded RNA about 21-25 nt in length.It is highly conserved and exhibit silencing-inducing effects at the post-transcriptional level of target genes,which regulate biological processes including growth and development,immune response.Vitamin D₃is an essential substance in fish diets.Because of its special production pathway,there is no way for fish to manufacture vitamin D by themselves and they must rely on exogenous additions to maintain their normal growth and development needs.Various studies have shown that fish are highly tolerant to vitamin D₃ and adding vitamin D₃ into feed can enhance the immunity and disease resistance of fish and reduce mortality when fighting off pathogenic microorganisms.However,whether mi RNAs in fish can respond to dietary vitamin D₃?Will the mi RNA responding to vitamin D₃ can regulates the immune response through the JAK-STAT immune pathway?In this study,we selected juvenile Pelteobagrus fulvidraco as the main subject.Then carried out a 12 weeks experiment on the nutritional regulation of vitamin D3 culture.Following the breeding experiment,an Edwardsiella ictaluir attack experiment was carried out.Followed by high-throughput sequencing of head,kidney and spleen tissue after the attack experiment,screening of differentially expressed mi RNAs with target gene bioinformatics prediction and enrichment analysis for differentially expressed mi RNAs and validation to explore the mechanism of vitamin D₃ mediated regulation of immune response by mi R-194a through JAK-STAT signaling pathway.The main results of the present study are as follows:1.Effect of different concentrations for dietary vitamin D₃ on mi RNATo investigate which mi RNAs responded to different concentrations of dietary vitamin D₃,we designed diet group 1 with a vitamin D₃ concentration of 1120 IU/kg,diet group 2 with a vitamin D₃ concentration of 3950 IU/kg,and diet group 3 with a vitamin D₃concentration of 16,600 IU/kg.Five co-occurring differentially expressed mi RNAs（mi R-194a,mi R-192,mi R-2188-3p,mi R-122,mi R-459-5p）were identified in the head kidney and spleen tissue of feed group 2 relative to feed group 1 after high-throughput sequencing,however,no co-occurring differentially expressed mi RNAs were found in the head kidney and spleen tissues of feed group 3 relative to feed group 1.For all differentially expressed mi RNAs,target genes were predicted and enriched,and the target genes of mi R-194a were found to be enriched in the JAK-STAT signaling pathway.Based on the above results,we predicted that the vitamin D₃ mediates the regulation of the immune response process by mi R-194a through the JAK-STAT signaling pathway,and validated in subsequent experiments.2.The validation of mi R-194a with its target genes related to the JAK-STAT pathwayThe mi RNA-target gene prediction software predicted the existence of potential binding sites for mi R-194a to the target genes jak2a and tyk2.For determining whether mi R-194a enables regulation of jak2a and tyk2,we constructed a wild-type（WT）recombinant plasmid by cloning the fragment of the jak2a and tyk2 binding site with the psi CHECK-2 plasmid,and a mutant recombinant plasmid（MUT）by mutating six bases of the binding site to the reverse complementary bases of the original bases.The fluorescence activity of the reporter genes was examined by co transfecting HEK-293T cells with mi RNA mimics,mi RNA NC together with the recombinant plasmid,then finally it was identified that mi R-194a has a targeted regulatory relationship with the target genes jak2a and tyk2,which can exert a suppressive effect on jak2a and tyk2.3.An investigation of the mechanism of vitamin D₃ mediated mi R-194a for immune response by the JAK-STAT signaling pathwayBy macrophage experiments,mi R-194a was also detected in cells in response to different concentrations of active vitamin D₃ within the culture medium as well as exerting the same inhibitory effect on the target genes of jak2a and tyk2.We found that with the activation of the JAK-STAT signaling pathway,the expression of anti-inflammatory response（AIR）factors showed an upward trend with the increase of vitamin D₃concentration in the culture medium（P<0.05）,the inflammatory pathway NF-κB pathway was inhibited（P<0.05）,and the regulation of macrophage transition from M1 type to M2type polarization（P<0.05）.In summary,the present study demonstrates that mi R-194a can respond to changes in vitamin D₃ in vivo and in vitro experiments,dietary vitamin D₃regulates the expression of the target genes jak2a and tyk2 by promoting the expression of mi R-194a.Moreover,indirect effects were exerted on downstream genes in the JAK-STAT pathway.Dietary vitamin D₃ mediated mi R-194a regulated the immune response of Pelteobagrus fulvidraco through the JAK-STAT signaling pathway.