The Transcription Factor DMyc Regulates The Molecular Mechanism Of Drosophila Imd Pathway Innate Immune Homeostasis

Drosophila melanogaster innate immunity mainly includes two signalling pathways:Toll andImd,which are highly similar to TLR?Toll-like Receptor?pathway andTNFR?Tumor Necrosis Factor Receptor?pathway in mammals.Therefore,Drosophila is an important model organism for the study of human innate immunity,which serves as a bridge for the study of the relationship between insects andhuman innate immunity.The systematic study on the mechanism of Drosophila innate immune response is not only helpful to reveal the maintenance mechanism of Drosophila immune homeostasis,but also has important scientific significance for the further study of mammalian immune regulation mechanism.The innate immunity of Drosophila is the first line of defense against the invasion of exotic pathogenic microorganisms.However,the over-activation or deficiency of immune response will cause varying degrees of damage to the organism.Therefore,in order to avoidthe imbalance of immune homeostasis,accurate activation andeffective inhibition of the expression of key factors in Drosophila Toll or Imdsignalling pathway is indispensable.At present,in addition to various activators andsuppressors,it has also been foundthat micro RNA?mi RNA?can participate in Drosophila immune response through targeting genes in the immune signalling pathway,but how is the expression of mi RNA involvedin immune regulation regulatedin the process of immune response?So far,it is still not clear,andfurther research is needed.As we all know,Myc is a very important transcription factor in organisms,which plays an important role in a variety of basic cellular activities,including cell cycle,cell growth,proliferation,apoptosis.However,whether Drosophila Myc?dMyc?is relatedto Drosophila innate immune response,andwhether dMyc can participate in Drosophila innate immune response by regulating the expression of one or more mi RNA?Therefore,aroundthese above important scientific issues,the following studies were carriedout:1.In order to determine the function of dMyc in Drosophila Imdimmune pathway,several dMyc mutant flies strains were constructedby Gal80^ts-tub-Gal4system:dMyc high-expressing flies strain,dMyc knock-down flies strain,anddMyc anddMyc-RNAi co-highexpressing flies strain.After infection with E.coli,the expression of Diptericin,the landmark antimicrobial peptide at the downstream of Drosophila Imdpathway,was detected.The results showedthat high expression of dMyc coulddown-regulate the expression of Diptericin,suggesting that it may negatively regulate the Drosophila Imdimmune response.2.By employing a variety of bioinformatics analysis methods,mi RNA that potentially relatedto Drosophila Imdimmune pathway andregulatedby dMyc was obtained.Targetscan andmi Randa software were usedto predict the target relationship between mi RNA and63 genes associatedwith Drosophila Imdpathway.Furthermore,mi RNAs potentially regulatedby dMyc was obtainedby using PROMO website andTransmi R v2.0 database.Intersecting with the differentially expressedmi RNA?DEmi RNA?obtainedby previous small RNA-seq in our laboratory,the results showedthat dMyc couldregulate 12 mi RNA?mi R-10,mi R-1012,mi R-184,mi R-275,mi R-276a,mi R-277,mi R-278,mi R-279,mi R-2b-2,mi R-965,mi R-996 andmi R-998?targeting immune-relatedgenes in the Drosophila Imdpathway.3.Through the immune correlation screening of high-expression mi RNA andthe positive correlation screening between the expression of dMyc andmi RNA,it was foundthat only mi R-277 regulatedby dMyc couldparticipate in the Drosophila Imdimmune response.Furthermore,the dual-luciferase reporter system was usedto analyze the promoter activity of mi R-277 gene.It was confirmedthat dMyc couldactivate the transcription of mi R-277 through directly binding with mi R-277 promoter region through Chromatin Immunoprecipitation-q PCR assay.4.Through bioinformatics analysis,it was foundthat the potential target genes of mi R-277 are two key signal factors in the Imdpathway,imdandTab2.The Dual-luciferase report experiment in Drosophila S2 cells further showedthat mi R-277coulddirectly target imdandTab2-Ra/b 3'UTR,but not Tab2-Rc 3'UTR.The results reveal the molecular mechanism that mi R-277 negatively regulates the Drosophila Imdimmune response by inhibiting the expression of imdandTab2-Ra/b.5.The dMyc andmi R-277 sponge?mi R-277 SP?co-highexpressing flies strain was constructedto explore the role of dMyc andmi R-277 in the Drosophila Imdimmune response.The results showedthat in the context of the level of mi R-277 in dMyc andmi R-277 sponge co-highexpressing flies returning to the normal level,the Diptericin,targets imdandTab2 were also recoveredto a certain extent.This indicates that dMyc can indeedinhibit the key immune signal factors imdandTab2 by activating mi R-277's transcription,andthen inhibit the expression of Diptericin,thus negatively regulating the Drosophila Imdimmune response.6.Through the study on the dynamic expression patterns of Diptericin,dmyc,mi R-277 andtargets imdandTab2 in wild-type flies infectedwith E.coli,it was foundthat dMyc playeda negative regulation function in the middle andlater stage of immune response,indicating that the negative regulation of dMyc plays an important role in the recovery of Drosophila immune homeostasis.In addition,the survival of dMyc high-expressing flies after infectedwith E.cloacae was further analyzed,andit was foundthat the high expression of dMyc improvedthe survival ability of Drosophila.The results of this paper indicate that dMyc plays a crucial role in maintaining Drosophila Imdimmune homeostasis.On the one hand,down-expresseddMyc coulddown-regulate mi R-277 expression to further up-regulate the expression of its target genes imdandTab2 at the early stage of E.coli infection,whilst up-regulatedimdandTab2 couldfurther promote the expression of Dpt to effectively resist the invasion of pathogenic bacteria.On the other hand,to prevent the overactivation of Imdimmune response,over-expressedAMP Dpt couldinduce the up-regulate expression of dMyc to improve the expression of mi R-277,whilst the up-regulatedmi R-277 coulddown-regulate the expression of imdandTab2 to further inhibit the expression of Dpt.In conclusion,the results of this paper reveal that the transcription factor dMyc plays an important regulatory role in Drosophila innate immune response,andis a key regulatory factor to prevent the overactivation of the immune response.The results of this study not only elucidatedthe new functions andregulatory modes of dMyc andmi R-277,but also providednew insights andideas for the deep revelation of the complex regulatory mechanisms of Drosophila innate immune homeostasis maintenance.