Molecular Mechanism Of MiR-190 Involved In The Regulation Of The Drosophila Imd Innate Immune Pathway

Drosophila melanogaster is an important model organism to study the mechanism of innate immunity regulation in biological research.Drosophila melanogaster have two important and classical innate immune signaling pathways: Imd and Toll.These two pathways have good homology with mammalian TNF-? and TLR signaling pathways,respectively.Therefore,the study on Drosophila melanogaster innate immunity provides a reference to further understand the regulatory mechanism of mammalian innate immunity.Studies have shown that microRNAs can be involved in the negative regulation of gene expression in post-transcriptional level,which play an important role in many physiological processes.Thus,exploring the function of microRNAs in the immune pathway is important for us to understand the negative regulation of the immune system in insects and mammals and it is of great significance to elaborate pathogenesis of mammalian immune diseases and study treatment methods.Combined with the previous work of our research group,through high-throughput sequencing and experiments,it was found that miR-190 can participate in the negative regulation of Drosophila melanogaster Imd signaling pathway.The research contents of this paper as follows:1.The expression of miR-190 in wild type Drosophila(w¹¹¹⁸)stimulated by Gramnegative bacteria Escherichia coli detected by real-time quantitative PCR(qRTPCR).The result showed that miR-190 is involved in innate immune response in Drosophila melanogaster.2.Use bioinformatics and microRNA prediction software,miRanda and Target Scan to predict the target genes of miR-190.Intersecting the results of two software predictions we found that miR-190 can target the coactivator Tab2 and immunityrelated gene akirin in the Imd pathway.3.Escherichia coli was used to stimulate Drosophila lines with high expression of miR-190,co-expression of miR-190 and miR-190 sponge,miR-190 KO Drosophila lines and w¹¹¹⁸.RNA was extracted at 0h,3h,6h,12 h,24h and 48 h respectively and then use the Real-time quantitative fluorescence technique(qRT-PCR)to quantitate the downstream antimicrobial peptides and target genes.Futher verify the immunocorrelation of miR-190 and the relation with Tab2 and akirin.The results indicated that miR-190 could inhabit gene expression of Tab2 and akirin to participate the regulation of innate immunity of Drosophila melanogaster;4.The plasmid pAc-miR-190,pAc-LUC-Tab2-RA/RB-3'UTR,pAc-LUC-Tab2-RC-3'UTR,pAc-LUC-akirin-RB/RC/RD/RF-3'UTR,pAc-LUC-akirin-RA/RE-3'UTR were constructed to verify the direct targeting relationship of miR-190 to the target gene in vitro.The result showed that miR-190 could target Tab2 directly;5.The survival rate of flies with high expression of miR-190 and miR-190 KO were observed after injection with Enterobacter cloacae that can cause death.Result showed that high expression of miR-190 incressed mortality and miR-190 KO lines showed lower mortality rate.This result futher demonstrated miR-190 play a important function in Drosophila melanogaster innate immunity.