Study On The Regulatory Role Of MiR-964 In The Immune Response Of Drosophila Toll

Drosophila melanogaster is a model organism that plays an important role in the study of innate immune mechanisms.The Toll pathway is one of the most important innate immune pathways in Drosophila,and it can resist the invasion of Gram-positive bacteria through a series of signal transduction.Recent studies have shown that miRNA play an important regulatory role in the process of immune signal transmission in Drosophila.miRNA is a kind of 21-24nts small non-coding RNAs which is widespread in the genome of animals and plants.By binding to specific sites of target mRNA,they inhibit mRNA translation or induce mRNA degradation,mediating many important physiological activities and processes regulate the expression of genes at the post-transcriptional level.In the previous work,we screened by high-throughput sequencing,and found miR-964 is significantly down-regulated after stimulated by Gram-positive bacteria,so miR-964 is likely to be an important regulatory factors involved in the Drosophila Toll pathway.In this study,the experiments in vitro and in vivo are confirmed that the miR-964 can regulate the Toll signaling pathway by directly targeting the Drs gene using the method of experimental biology and bioinformatics.The main experimental results obtained in this paper are as follows:1.Useing septic injury with Gram-positive bacteria(E.faecalis)to examine the effect of miR-964 on the fly's survival.The miR-964 overexpressing flies(Gal80ts;Tub>miR-964)showed a statistically significant reduction in survival compared with both the control flies(w1118)and miR-964 knockout flies(miR-964 KO).However,there was no difference between the miR-964 knockout flies(miR-964 KO)and control flies(w1118).2.The expression level of gene miR-964 in wild-type flies(w1118)was significantly increased at 24h,48h and 72h after injection with M.luteus.and the antibacterial peptide Drs expression in miR-964 overexpressing flies(Gal80ts;Tub>miR-964)was significantly lower than control flies(Gal80ts;Tub-gal4)at 24h post-infection.Whereas,the antibacterial peptide Drs expression in miR-964 knockout flies(miR-964 KO)was significantly higher than wide-type flies(w1118)both at 12h and 24h post-infection.We overexpressed miR-964 and a miR-964 sponge in adult flies and found that the antibacterial peptide Drs was restored by qRT-PCR and Drs-green fluorescent protein(GFP)reporter.Experiments show that miR-964 can respond to the stimulation of Gram-positive bacteria and affect the expression of the antibacterial peptide Drs in the Toll pathway.3.To understand the molecular mechanism underlying the action of miR-964 in the Toll-mediated innate immune pathway,we carried an in silico genome-wide screening in Drosophila using three different online miRNA target prediction methods(TargetScan,miRanda and PITA).We finally identified Spz and Drs two Toll signaling pathway-associated genes.4.We performed luciferase reporter assays in Drosophila S2 cells.This result indicates that the Drs not Spz is a target of miR-964.5.We further determine that Drs is the direct target of miR-964 by qRT-PCR and Western Blotting in vivo.In this paper,we find that miR-964 can negatively regulate the Toll signaling pathway by directly targeting the 3' UTR of the gene Drs and control the expression of the antibacterial peptide Drs to participate in the innate immune regulation of fruit flies.It provides a reference for studying the regulation of miRNA in fruit fly immune pathway.