Study On The Evolution Of MiRNA Clusters In Grass Carp And The Mechanisms Of MiR-n3 Involved In Resistance To Bacterial Infection

Grass carp(Ctenopharyngodon idella)is an important freshwater fish that is widely farmed worldwide.In freshwater fish farming,economic losses due to diseases are one of the main factors affecting the development of the aquaculture.Among them,septicemia induced by Aeromonas hydrophila infection has become one of the common diseases in grass carp.Sepsis is an inflammatory response syndrome that occurs when the organism is infected.Many studies have shown that miRNAs play an important role in the antimicrobial immune response,and multiple miRNAs have been shown to be involved in the inflammatory response in grass carp.In animals,miRNAs can be expressed synergistically in clusters.In this paper,we analyzed the clustering pattern,evolutionary characteristics and expression of grass carp miRNAs,and specifically investigated the mechanism by which lncRNA-adm2 can regulate the inflammatory response induced by Aeromonas hydrophila through binding miR-n3,and miR-n3 targets adm2 to regulate the inflammatory response,and the main findings of this paper are shown below:1.Clustering and evolutionary characterization of miRNAsIn this study,a total of 1,513 miRNAs were identified by small RNA sequencing.By comparing with other Teleost fish miRNAs,1,442 of them were found to be unique to grass carp and 71 were conserved miRNAs.397 miRNAs were found to be less than10 kb apart and clustered into 171 miRNA clusters by calculating the distance between adjacent miRNAs.The clusters were traced back and found that the grass carp miRNA clusters were mainly formed through de novo generation.The analysis of specific and common miRNAs in the miRNA clusters showed that common miRNAs were significantly enriched in the clusters.Calculation of miRNA evolution rates revealed that clustered miRNAs were more conserved and evolved more slowly,while specific miRNAs usually evolved at a higher rate.Tissue specificity analysis of all miRNAs in grass carp showed that grass carp-specific miRNAs were more tissue specific than common miRNAs.The expression pattern of grass carp-specific miRNAs was carefully studied,and the expression of grass carp-specific miRNAs was generally lower than that of common miRNAs in all tissues.miRNA cluster co-expression analysis using WGCNA revealed that clustered miRNAs were clustered in the same modules,and the expression levels were strongly correlated.2.miR-n3 regulation of inflammatory responsemiR-n3,which was expressed in all tissues,was selected for follow-up study,and it was found that miR-n3 reached the highest expression level in spleen tissue.The expression of miR-n3 fluctuated irregularly after Aeromonas hydrophila infection in grass carp,suggesting the involvement of miR-n3 in the immune response against bacterial infection in grass carp.Adm2 was predicted to be a potential target gene of miR-n3 using RNAhybrid software.The results showed that overexpression of miR-n3 decreased the expression of adm2.On the contrary,the expression level of adm2 was up-regulated after inhibition.Next,a dual luciferase reporter system was used to confirm that miR-n3 targets adm2 to act.The expression levels of the downstream pro-inflammatory factors(il-1β and il-6)were significantly upregulated after overexpression of miR-n3,and the expression of the anti-inflammatory factor il-10 was significantly reduced after overexpression of miR-n3.The present results contribute to further understanding of the regulatory role of miR-n3 on the inflammatory response of grass carp.3.Interaction of miR-n3 with lncRNA-adm2Initially,lncRNA-adm2 was predicted to bind to miR-n3 by RNAhybrid software.After Aeromonas hydrophila infection of grass carp,lncRNA-adm2 expression levels showed significant fluctuations relative to PBS-injected grass carp tissues.Overexpression of lncRNA-adm2 significantly suppressed the expression of miR-n3;the expression level of miR-n3 was significantly up-regulated after inhibition of lncRNA-adm2.The dual luciferase reporter system confirmed that overexpression of miR-n3 significantly reduced the luciferase activity of lncRNA-adm2,indicating that miR-n3 can bind to lncRNA-adm2.Inhibition of lncRNA-adm2 increased the expression of downstream pro-inflammatory factors(il-1β and il-6)and decreased the expression level of anti-inflammatory factor il-10.The above data suggest that lncRNA-adm2 can directly interact with miR-n3 in the immune response against bacterial infection in grass carp.