Immune Regulation Of Gga-miRNA-181a-5p Mediated CGAS-STING Pathway On FAdV-4 Infection

Fowl Adenovirus Serotype 4(FAdV-4)is a non enveloped ds DNA virus.In recent years,Hepatitis-hydropericardium syndrome(HHS)caused by FAdV-4 has been widely prevalent,which has caused great economic losses to poultry industry in China and even the world,and has become a serious cause of damage to poultry in China,next only to avian influenza virus.Micro RNA plays an important regulatory role in the process of virus host interaction.It is widely involved in the process of virus infection and bidirectional regulation of host innate immune response.However,the role and mechanism of miRNA in the host cell response caused by FAdV-4 infection is still unclear,which needs to be further studied.Cyclic GMP-AMP synthase(cGAS)-interferon stimulating factor(STING)signaling pathway is an important part of the innate immune system.It plays an important role in the DNA virus immune response.Its function is to detect the presence of cytoplasmic DNA and trigger the expression of type I interferon and inflammatory genes in the response.The interaction between cGAS-STING signaling pathway and miRNA is a multi-directional process.The recognition of viral infection by cGAS-STING signaling pathway will start the signal transduction cascade of innate immune response,resulting in the change of miRNA expression.At the same time,miRNA can also regulate gene expression and regulate innate immune response,but how miRNA plays a regulatory role between FAdV-4infection and cGAS-STING pathway has not been reported.Therefore,it is of great significance to study the mechanism of regulating FAdV-4 infection by cGAS-STING pathway mediated by miRNA.In this study,we established a model of FAdV-4 infection with LMH cells infected by FAdV-4,obtained the miRNAs expression profile after FAdV-4 infection by high-throughput sequencing technology,screened the differentially expressed miRNAs and interferon signaling pathways involved in FAdV-4 infection with LMH cells,and conducted in-depth research the immune response induced by FAdV-4infection and its related regulatory mechanism provide new strategies for elucidating the interaction between FAdV-4 infection and host innate immunity,clarifying the molecular pathogenesis of FAdV-4 infection and screening new therapeutic targets against FAdV-4infection,the main research content and results are as follows:1.Establishment of FAdV-4 cell infection model and screening of its miRNA expression profile and interferon pathwayIn order to explore the biological function of miRNA in the process of FAdV-4 infection,the dynamic changes of host miRNA and its effect on host innate immunity were revealed from the molecular and cellular level.By establishing the cell model of FAdV-4 infection,the interaction between FAdV-4 and host was studied.LMH cells were inoculated with 1MOI FAdV-4 AH-F19 strain.Cell samples were collected at 0 h,12 h,24 h,48 h and 72 h after infection for sRNA sequencing analysis.The results showed that a total of 2861 miRNAs were obtained from all samples,including 962 known miRNAs and 1899 new predicted miRNAs.There were 176,174,383 and 688 differentially expressed genes at 12 h,24 h,48 h and 72 h after infection,respectively.The target genes of different miRNAs were predicted,and the target genes were analyzed by GO and KEGG.The results showed that cGAS-STING signaling pathway,MAPK signaling pathway and m TOR signaling pathway were involved in the process of FAdV-4 infection.The expression of chSTING was up-regulated at 12 h after infection.However,the expression of chSTING was significantly inhibited at 24 and 48 h,and the expression of IFN-β was also inhibited.In this study,we obtained the miRNA expression profile of LMH cells infected with FAdV-4,and screened chSTING to carry out the follow-up study,so as to provide data for the mechanism of FAdV-4 infection and its interaction with the host.2.The role of chSTING in FAdV-4 infection-induced IFN-β production and virus replicationThe expression of chSTING was significantly affected by FAdV-4 infection.In order to explore the role of chSTING in the immune response between FAdV-4 infection and host cell virus,the monoclonal antibody against chSTING was prepared and the class and subtype of the antibody were identified.Then,the stable overexpression cell line of chSTING was constructed by lentivirus technology,and the chSTING deficient cell line was constructed by CRISPR / Cas9 technology.Then the wild LMH cell line,chSTING overexpression cell line and chSTING-KO cell line were infected with FAdV-4.Finally,detected the expression levels of IFN-β,IL-1 β,IL-8 and some ISGS and the copy number of FAdV-4.The results showed that two hybridoma cell lines named 3c6 and 5B9 could stably secrete anti chSTING antibody.The light chain type of the two Mc Abs was kappa type,and the heavy chain type was Ig G1.chSTING can induce IFN-β,IL-6 and IL-8,increase the expression of gpatch-3 and MX1,and inhibit the replication of FAdV-4;on the contrary,chSTING can inhibit the expression of IFN-β and other cytokines,GPATCH-3and MX1,and promote the replication of FAdV-4.Therefore,chSTING plays a positive role in regulating the expression of IFN-β and some ISGS during FAdV-4 infection.This study further confirmed that cGAS-STING signaling pathway plays a key role in the induction of IFN-β in LMH cells infected with FAdV-4.The role of chSTING in the pathogenesis of FAdV-4 and the prevention and control of FAdV-4 were explored.3.Effect of gga-miRNA-181a-5p targeting chSTING on FAdV-4 infectionMi RNAs play an important role in the interaction between virus and host.The results of previous studies showed that the expression of chSTING was significantly inhibited in the late stage of FAdV-4 infection,but it is not clear how miRNA regulates chSTING.Based on the results of this study,the regulatory effect of miRNA on chSTING in FAdV-4infection was studied.Firstly,miRNA of chSTING 3 ’UTR was predicted by miRBase;Then,detect the specific binding miRNA with chSTING and its effect on chSTING expression by double luciferase reporter gene system,Western blot and IFA.Finally,the effects of target miRNA on the replication of FAdV-4 and the signaling pathway mediated by chSTING were explored.The results showed that gga-miR-181a-5p and gga-miR-12286-5p were screened by bioinformatics analysis,but gga-miR-181a-5p could regulate the activity of chSTING 3 ’UTR,and negatively regulate the expression of chSTING at m RNA and protein levels.Overexpression of gga-miR-181a-5p can inhibit the replication of FAdV-4 in LMH cells,on the contrary,it can promote virus replication;gga-miR-181a-5p can inhibit the signaling pathway mediated by chSTING,and negatively regulate NF-κB,IRF7 and IFN-β signaling and some antiviral gene expression.This study proves that gga-miR-181a-5p is a negative feedback regulator in the study of antiviral immune response,which provides a basis for exploring the interaction between FAdV-4and host natural immune and potential anti viral targets.In conclusion,the miRNA expression profile of LMH cells infected with FAdV-4 was analyzed to screen the IFN pathway activated by infection.It was further found that in the early stage of FAdV-4 infection,the expression of chSTING was significantly increased,and its expression was significantly inhibited in the later stage of infection.In order to explore the role of chSTING in IFN β production and virus replication induced by FAdV-4infection,the expression of IFN-β and some ISGS was regulated by chSTING in the transcription and protein levels.gga-miR-181a-5p was found to inhibit the signaling pathway mediated by chSTING for the first time.FAdV-4 can regulate the host natural immunity through gga-miR-181a-5p/chSTING signal axis to achieve FAdV-4 mediated immune escape,enrich the theoretical basis of FAdV-4 infection and pathogenesis,and lay a foundation for further study on the pathogenesis of FAdV-4.