Study On The Regulation Of Inflammatory Factors In Ctenopharyngodon Idellus MiR-21 And MiR-142a-3p During Bacterial Infection

Grass carp(Ctenopharyngodon idellus)can cause severe sepsis after being infected by Aeromonas hydrophila.In the early stage of our research group,nine key miRNAs were identified in grass carp susceptible and disease-resistant populations.This paper conducted follow-up functional analysis on miR-21 and miR-142a-3p.We found that grass carp miR-21 can regulate the inflammatory response induced by A.hydrophila by targeting jnk and ccr7,and miR-142a-3p can regulate the inflammation response caused by A.hydrophila infection by targeting tnfaip2 and glut3.The main findings are as follows:1.Grass carp miR-21 showed a downward trend during the infection of A.hydrophila and LPS(endotoxin of Gram-negative bacteria).The target genes of miR-21,jnk and ccr7,were predicted by biological information software.miR-21 was negatively correlated with the expression patterns of jnk and ccr7 during the infection of A.hydrophila.Overexpression of miR-21 can significantly inhibit the expression of jnk and ccr7;after miR-21 inhibition,the expression level of jnk and ccr7 increased significantly.The dual luciferase reporter system further confirmed the miR-21 targeted regulation of jnk and ccr7.miR-21 inhibits downstream pro-inflammatory factors,tnf-α,il-1β,il-6,and il-12 by targeting jnk and ccr7.2.The potential target genes tnfaip2 and glut3 of miR-142a-3p were predicted by biological information software.During the infection of A.hydrophila,the expression of tnfaip2 and glut3 were both increased,which showed a negative correlation with miR-142a-3p.When miR-142a-3p was overexpressed,tnfaip2 and glut3 were significantly inhibited,and knocking down miR-142a-3p increased the expression of tnfaip2 and glut3;the dual luciferase reporter system further confirmed the targeted regulation of miR-142a-3p on tnfaip2 and glut3.miR-142a-3p targets tnfaip2 and glut3 to regulate the expression of downstream pro-inflammatory factors(tnf-α,il-1β and il-8);and promotes the polarization of Kupffer cells(grass carp liver macrophages)to M2 type.The results of CCK-8 and Caspase-Glo3/7 showed that inhibition of miR-142a-3p promoted cell survival,and overexpression of miR-142a-3p promoted apoptosis,indicating that miR-142a-3p can target tnfaip2 and glut3 to induce apoptosis.3.Identified 4 JNK and 6 p38 genes from the grass carp genome database,including,which were distributed in 7 of 24 chromosomes.Analysis of protein conserved domains revealed that they all contain double phosphorylation sites(JNKs: Thr-Pro-Tyr,p38: Thr-Gly-Tyr).The expression of JNK and p38 family genes are low under normal conditions.After bacterial infection,the expression levels of jnk3,p38 beta,p38 gamma a and p38 gamma b were significantly increased,indicating that they have a potential role in the immune response after bacterial infection.