Drosophila Melanogaster MiR-1000 Negatively Regulates Toll Signaling By Targeting Myd88

In Drosophila melanogaster,the humoral immunity composed of antimicrobial peptides and reactive oxygen species plays an important role in defending the invading pathogens.The Toll pathway and IMD pathway regulate the humoral immunity,which are highly conserved.The Toll pathway in Drosophila is homologous to the Toll-like Receptor(TLR)pathway in mammals,while the Imd pathway is homologous to the TNF-? pathway.In recent years,although the main components of these two pathways have been identified,little is known about how immunity is precisely regulated.Imbalance of immunity homeostasis is usually harmful to the organism.For example,the continuous activation of immunity can reduce the life span and survival rate of fruit flies.Therefore,the systematic study of the regulation mechanism of immune pathway in Drosophila melanogaster not only contributes to the understanding of immune homeostasis regulation,but also provides a new insight into for the study of mammalian immune regulation mechanism.In this study,we found that miR-1000 in wild type flies is upregulated after infecting Micrococcus luteus.Then we construct highly expressing flies and miR-1000 KO flies.After infecting miR-1000 highly expressing flies and miR-1000 KO flies with Micrococcus luteus,we found that the expression level of Drs(Drosomycin),a downstream gene of Toll signaling pathway,was significantly decreased in miR-1000 highly expressing flies than the control flies,while expression level of Drs in miR-1000 KO flies was significantly increased,indicating that miR-1000 was indeed a negative regulator of the Toll signaling pathway.Then,we used bioinformatics methods to predict a number of candidate target genes of miR-1000,including mod SP and Myd88,which play important roles in the Toll signaling pathway.After that,we detected the expression of Myd88 and mod SP in miR-1000 highly expressing flies and miR-1000 KO flies after infection with Micrococcus luteus.It was found that when miR-1000 was highly expressed,both Myd88 and mod SP were down-regulated,while in miR-1000 KO flies,the expression of Myd88 was increased,but the expression of Mod SP was not significantly changed.Then we used the dual fluorescence reporting system in S2 cell line to explore the relationship between miR-1000 and mod SP as well as Myd88,and found that miR-1000 could down-regulate the expression of the reporter gene with the 3? UTR of Myd88,but could not bind the 3? UTR of mod SP.After mutating the binding site of miR-1000 in Myd88 3? UTR,the expression of the reporter gene have no significant difference with the control,indicating that Myd88 is a direct target gene of miR-1000.In order to further prove the negative regulatory role of miR-1000 in the Toll signaling pathway,we used the miR-1000 sponge flies to construct a miR-1000 & miR-1000 sponge co-expressed flies,which could compromise the high expression of miR-1000.It was shown that the expression of Myd88 and Drs were keeping with the expression of miR-1000,which further proved that miR-1000 negatively regulated the Toll signaling pathway by targeting Myd88.In order to investigate the function of miR-1000 in Drosophila immunity,Enterococcus faecalis was used to infect miR-1000 highly expressing flies and miR-1000 KO flies.It was found that the survival rate of miR-1000 highly expressing flies decreased,while the survival rate of miR-1000 KO flies increased,indicating that the ectopic expression of miR-1000 can disrupt the immune homeostasis.In conclusion,miR-1000 inhibits the expression of Myd88,thus inhibiting the Drosophila Toll pathway and the expression of AMPs regulated by Toll pathway.