Study On The Regulatory Role Of MiR-977 In The Toll Immune Pathway Of Drosophila

Non-coding RNA,especially microRNA(miRNA),plays an extremely important role in various life processes and has been a hot research field in life sciences.The ability of miRNAs to regulate gene expression at the post-transcriptional level plays a key role in maintaining homeostasis in vivo.It is well known that excessive activation or deficiency of immune response can cause different degrees of damage or even disease in organisms.Therefore,it is of great theoretical and practical significance to study the function and regulation mechanism of miRNA in animal immune homeostasis.Drosophila melanogaster has become an important model organism for studying immune function and immune response mechanism of animals because of its easy feeding,short growth cycle,easy to distinguish male and female,complete genome sequence and rich genetic resources.The innate immunity of Drosophila mainly resists the infection of pathogenic microorganisms by activating the Toll signaling pathway and the Imd signaling pathway to produce antimicrobial peptides(AMP),while the Drosophila Toll signaling pathway and the Imd signaling pathway are also associated with mammalian organisms.The TLR pathway is homologous to the TNF-? signaling pathway,and they share the same evolutionary origin in the regulation of immune responses.Therefore,an in-depth study of the regulatory mechanisms of miRNAs in Drosophila Toll and Imd signaling pathways is important not only to clearly elucidate the function and mechanism of miRNAs in Drosophila immune homeostasis,but also to further reveal miRNAs in insects.The experimental results can provide important insights for innate immunity and the regulatory mechanisms in human.To this end,this paper has carried out a series of studies on how to regulate the scientific response of miRNA to Toll signal immune response,and obtained the following main results:1.The overexpression drosophila of miR-975/976/977 was infected with Gram-positive bacteria Micrococcus luteus(M.luteus),and then real-time fluorescence quantitative(qRT-PCR)was used to detect the expression of antibacterial peptide(Drosomycin,Drs)in Toll signal pathway at 6 time points after infection.The results showed that miR-975/976/977 overexpression flies,which significantly decreased the level of Drs at 24 h and 48 h after infection compared with control.The same experimental method was used to determine the Drs of the gene-deficient Drosophila miR-975/976/977 KO.It was found that the content of Drs in knockout miR-975/976/977 KO drosophila was significantly up-regulated at 12 h,24h and 48 h compared with control.All the results indicated that miR-975/976/977 can down-regulate the Toll signaling pathway;2.To determine which miRNA of miR-975/976/977 regulate the expression of Drs in Drosophila,we further tested the level of miR-975,miR-976 and miR-977 overflies at 6 times after infection by qRT-PCR.The expression of Drs in vivo showed that only the outcome of miR-977 was consistent with miR-975/976/977 clusters,which could negatively regulate the expression of Drs in Drosophila;3.To further reveal the mechanism to negatively regulate Toll signaling by miR-977,we used miRanda and TargetScan programs to predict the target genes that miR-977 may target.After the intersection,we found that miR-977 can target the Toll,myd88 and Dif in the Drosophila Toll signaling pathway.4.The expression levels of Toll,myd88 and Dif were detected by qRT-PCR.According to the quantitative results,miR-977 could only target Dif gene.5.Based on the analysis of the dual luciferase reporter system of miR-977,it was found that the immunoregulatory function miR-977,which targets the downstream three transcripts of Dif-RA/RB/RD to negatively regulate Drs expression in Drosophila Toll signaling pathway;In conclusion,this study found that miR-977 is a novel negative regulator involved in the negative regulation of Drosophila Toll signaling immune response,and revealed that miR-977 negatively regulates the expression of Drs by regulating three transcripts of Dif-RA/RB/RD.At the same point,the study also clarified that miRNA plays an important regulatory role in the innate immune response mediated by Gram-positive bacteria in Drosophila.