The Mechanism Of Grass Carp MiR-451 And Let-7b Regulating Inflammatory Response In The Process Of A.hydrophila Infection

Grass carp(Ctenophthanodon idella)is one of the important freshwater economically cultivated fish in China.During the cultivation process,it usually resists stress by enhancing a specific metabolic process.Aeromonas hydrophila is a gramnegative aquatic bacteria,which can easily induce bacterial septicemia after infecting grass carp.Sepsis is probably the most common disease in grass carp,which causes huge economic losses every year.Bacterial septicemia can lead to uncontrolled inflammatory reaction,which is the fundamental reason for the increase of incidence rate and mortality of grass carp.Starting from the lnc RNA-mi RNA-m RNA regulatory network,this study studied the influence of the negative feedback formed by the lnc RNAs and mi R-451 and let-7b of grass carp infected by A.hydrophila on the downstream target gene expression,and also analyzed the sequence characteristics of the gene family of the target gene slc4 a of let-7b.The main results of this paper are as follows:1.Lnc RNA-ANAPC2 and lnc RNA-NEFM combine with mi R-451 to regulate the inflammatory response after bacterial infectionBased on the previous research results of the research group,the co-location and co-expression screening were carried out according to the upstream and downstream5 K of the target gene.Lnc RNAs that may involve bacterial infection were selected:lnc RNA-ANAPC2 and lnc RNA-NEFM.After A.hydrophila infected grass carp kidney cells,lnc RNA-ANAPC2 and lnc RNA-NEFM and mi R-451 showed fluctuating expression patterns.The dual luciferase reporting system confirmed that lnc RNA-ANAPC2 and lnc RNA-NEFM could bind to mi R-451.Co-transfection of lnc RNA-ANAPC2 si RNA,lnc RNA-NEFM si RNA and control into CIK cells can reduce the expression of pro-inflammatory factors il-6,il-8,il-12 and il-1β.2.Lnc-hw/let-7b/hbegf/wnk4 a axis participates in the immune regulation of grass carp after bacterial infectionAfter the grass carp was infected by Aeromonas hydrophila,the expression patterns of lnc-hw and let-7b in the grass carp tissues showed fluctuations.The dual luciferase reporting system confirmed that lnc-hw could bind with let-7b.Overexpression of lnc-hw could significantly inhibit the expression of let-7b;After inhibiting lnc-hw,the expression level of let-7b increased significantly.The potential binding sites of let-7b with hbegf and wnk4 a were predicted by RNAhybrid.The double luciferase report system confirmed that hbegf and wnk4 a were the target genes of let-7b.In grass carp CIK cells,lnc-hw can affect the expression of downstream inflammatory factors il-6,il-8 and il-10 by binding let-7b.Lnc-hw can also transform let-7b to NF-κB Inhibitory effect of reporter gene luciferase activity.NF-κB The activation of the signal pathway requires energy.Based on the above results,we further discussed the effect of lnc-hw combined with let-7b on the glucose metabolism of grass carp macrophages and the survival of grass carp CIK cells,and concluded that lnc-hw can combine with let-7b macrophage glucose metabolism and the survival of grass carp CIK cells.3.Analysis of slc4 a gene familyThis paper also predicted another potential target gene slc4a4 a of let-7b,and screened genes with similar structure and function in the whole genome of grass carp,and defined them as the grass carp slc4 a gene family.There are 13 members of grass carp slc4 a gene family,which are distributed on 11 chromosomes.In the healthy tissues of grass carp,slc4a2 a and slc4a2 b are highly expressed in almost all tissues compared with other family members;After infection,the genes that were originally highly expressed in different tissues increased and decreased.Through the analysis of protein-protein interaction,it is concluded that the relationship between grass carp slc4 a gene family depends on the genes outside the family to obtain the connection,and the internal environment of grass carp is maintained by the complex regulatory network.