Identification And Function Study Of Differentially Expressed Genes In The Bursa Of Fabricius With Immunosuppression Induced By Dexamethasone

Stress is a self-protective physiological response induced by the body when it resists adverse stressor stimuli.At this time,in order to prevent the harm caused by stimulation,the animal body actively adapts to environmental changes and maintains the body’s steady state balance.In livestock and poultry production,moderate stress can continuously adjust and optimize the immune response capacity of the body,but the stress that exceeds the body’s burden will affect the body’s internal homeostasis,affect the growth and development of the body,and destroy the immune response The response system poses a great threat to the health of livestock and poultry,and seriously affects the economic benefits of the breeding industry.Based on the dexamethasone-induced immunosuppressive model we successfully constructed in the early study.RNA-seq technology was used to identify the expression patterns of miRNA and mRNA in the bursal organs before and after dexamethasone treatment.And then performed the correlation analysis of miRNA,mRNA and target genes to determine the significantly down-regulated miRNA in the bursal tissue treated with dexamethasone:gga-miR-146a-5p,which is the miRNA for subsequent functional analysis.The target genes of gga-miR-146a-5p were comprehensively predicted by bioinformatics software,combined with the results of gene function annotation,and the candidate target gene IRAK2 related to the immune response was screened and determined.Secondly,this experiment explored the effect of dexamethasone on the expression pattern of gga-miR-146a-5p at the cellular level,and verified the effect of gga-miR-146a-5p of immune response by in vitro transfection and co-treatment with dexamethasone and lipopolysaccharide.Then,using bioinformatics to predict the binding site of gga-miR-146a-5p and the candidate target gene IRAK2,construct the wild-type binding site of gga-miR-146a-5p and the candidate target gene IRAK2 vector and mutant vector,through the dual luciferase reporter system verifying the target relationship between gga-miR-146a-5p and IRAK2;After interference with IRAK2 expression in HD11 cell line,the expression of immune-related cytokines were detected,and the role of IRAK2 in the immune response process was explored.The main test results are as follows:1.A total of 88 significantly differentially expressed miRNAs and 935 significantly different mRNAs were identified,and candidate miRNAs(gga-miR-146a-5p)involved in the immune regulation of poultry bursal were screened.2.Dexamethasone treatment of HD 11 cells significantly inhibited the expression of gga-miR-146a-5p;3.CCK-8,EDU,cycle and apoptosis results show that gga-miR-146a-5p can promote the proliferation of HD11 cells and inhibit cell apoptosis;4.Gga-miR-146a-5p and dexamethasone co-treatment test showed that miR-146a-5p can increase the immunosuppressive effect of dexamethasone on macrophages;5.Gga-miR-146a-5p and lipopolysaccharide co-treatment test showed that miR-146a5p can antagonize the inflammatory response of macrophages induced by LPS;6.The result of dual luciferase report shows that IRAK2 is the target gene of gga-miR146a-5p;7.Interference with the expression of IRAK2 in HD11 cells can significantly inhibit the expression of immune-related cytokines;In summary,compared with the blank control group,the expression of gga-miR146a-5p in the dexamethasone treatment group was significantly down-regulated;ggamiR-146a-5p can significantly reduce immune-related cytokines in the chicken HD 11 cell line.Gga-miR-146a-5p can target the IRAK2 and inhibit its expression;IRAK2 significantly inhibits the expression of intracellular immune-related factors.It can be seen that gga-miR-146a-5p may regulate immune response by inhibiting the expression of IRAK2 gene.