Expression And Mechanism Of MiRNA Influenced By Stress-induced Immunosuppression And Immune Response

Stress-induced immunosuppression is one of the serious problems faced by the poultry industry,which often leads to the problems of poor immune effect or failure of vaccines,especially for chicken.Mi RNAs play an important role in immune regulation,which directly or indirectly affect the expressional activities of immune-related genes and participate in the regulation of immune function.However,the molecular regulation mechanism of circulating miRNAs in the process of stress-induced immunosuppression in poultry is still unclear.In this study,chicken was used as model animal,and immunized with avian influenza virus(AIV)vaccine under the conditions of normal body and dexamethasone(Dex)-induced immunosuppression.Serum,heart,liver,bursa of fabric,spleen,glandular stomach and other tissues were collected at ten time points of 1dpi(day post immunizaton),2dpi,3dpi,4dpi,5dpi,7dpi,14 dpi,21dpi,28 dpi and35dpi.Fluorescence quantitative stem-loop RT-PCR was used to analyze the expressional characteristics and change rules of 15 AIV-related serum circulating miRNAs,three significant differentially expressed miRNAs(miR-214,miR-24-3p and miR-20a-5p)were screened out,the tissue sources of them were identified,and the mechanisms of these candidate miRNAs were predicted using bioinformatics method.The results of the study were as follows:Firstly,in normal chickens immunized with AIV vaccine,10 AIV-related circulating miRNAs were significantly down-regulated or up-regulated at 2dpi,5dpi,21 dpi and 35dpi;In Dex-induced stress immunosuppressive chickens,the four time points of 2dpi,5dpi,14 dpi and 28 dpi were the time points with significant changes of circulating miRNAs;In stress-induced immunosuppressive chickens immunized with AIV vaccine,2dpi,5dpi,7dpi,14 dpi,21dpi and 28 dpi were the time points with significant changes in the amplitude and frequency of miRNAs fluctuations.Comparative analysis found that differentially expressed circulating miR-214,miR-24-3p and miR-20a-5p had better representation,so the three miRNAs were used for follow-up research.Secondly,the 11 tissue expression profiles of miR-214,miR-24-3p and miR-20a-5p under different conditions were identified,and the results showed that the expressional activities of the three miRNAs were significantly different in different tissues,states and stages.Lung,proventriculus,bursa of Fabricius,cecal tonsil,thymus,spleen,small intestine and colon might be the key tissues that regulated the differential expression of serum circulating miR-214 and miR-24-3p;Provencal stomach,bursa of Fabricius,thymus,spleen,small intestine and colon might be the major tissues that affected the differential expression of serum circulating miR-20a-5p.Interestingly,under the dual conditions of stress-induced immunosuppression and AIV vaccine immune response,the mechanisms of miRNAs might become more complicated,indicating that the functions and mechanisms of miRNAs might be different in different states.Finally,through bioinformatic analysis of miR-214,miR-24-3p and miR-20a-5p,it was found that the target genes of miR-214 were mainly enriched in pathways such as positive regulation of RNA polymerase II promoter transcription and MAPK.We speculated that miR-214 participated in immune regulation through the "miR-214-MAP3K3" and "miR-214-PTEN" pathways;The target genes of miR-24-3p were also mainly involved in the RNA polymerase II promoter transcription,MAPK and other pathways,and "miR-24-3p-RAP1B" and "miR-24-3p-RAP1A" might be the key pathways in immune regulation of miR-24;The target genes of 20a-5p were mainly involved in transcriptional regulation,autophagy,m TOR and Er Bb signaling pathways,and "miR-20a-5p-AKT3","miR-20a-5p-PTEN" and "miR-20a-5p-PIK3R1" might be important pathways.In this study,the expression characteristics of AIV-related serum circulating miRNAs were preliminarily explored,the differentially expressed miRNAs(miR-214,miR-24-3p and miR-20a-5p)were screened out from the immune response of AIV vaccines under stress-induced immunosuppressive condition,the key tissues(proventriculus,bursa of Fabricius,thymus,spleen,small intestine,colon)regulating the candidate serum circulating miRNAs under different conditions were found,and several signal pathways that miRNA participates in immune regulation were predicted.The results can provide theoretical and practical references for in-depth study of the molecular mechanism of stress-induced immunosuppression affecting immune response,and developing supplementary detection technology for poultry immune status using circulating miRNAs as the targets.