Study On The Dynamic TF-miRNA-lncRNA-gene Regulatory Network Of Drosophila Immune Response To The Stimulation Of Micrococcus Luteus

With the advanced study of non-coding RNA,it is found that various non-coding RNAs play very key roles in immune responses and several other biological processes,especially microRNA(miRNA)and long noncoding RNA(lncRNA).The homeostasis of the immune system is an important guarantee for organisms which needs precise control,because that both the excessive activation and insufficiency of the immune response will damage biological individuals.More and more studies have shown that both miRNA and lncRNA play very important roles in the regulation of immune homeostasis.However,the mechanism of how transcription factor(TF),lncRNA,miRNA and gene coordinate the precise regulation of immune homeostasis is still unclear,which needs further study.Drosophila melanogaster is a classical model organism,the convenient genetic operability,complete genome annotations and abundant mutant strains provide an important basis for studying the regulatory mechanisms of innate immune responses in Drosophila.In particular,Drosophila Toll and Imd signaling pathways are highly homologous to mammalian TLR and TNF-a signaling pathway respectively,revealing that Drosophila and mammals share a common evolutionary origin in innate immune regulatory pathways.Therefore,systematically studying the function and mechanism of the "TF-miRNA-lncRNA-gene" regulatory network in Drosophila innate immune responses will contribute to elucidate the maintenance mechanism of Drosophila immune homeostasis,and it has also an important scientific significance for further understanding the regulatory mechanism of mammalian innate immune response.For this,the present paper mainly focused on the important scientific issue of the mechanism of the " TF-miRNA-lncRNA-gene " regulatory network in innate immune response of Drosophila.We further performed RNA-seq and small RNA-seq from matched PBS-infected and Micrococcus luteus infected Drosophila w1118 adult males at three time points(3,12 and 24 h),respectively.The IncRNA and miRNA prediction flow were constructed base on RNA and small RNA sequencing data,and new potential lncRNAs and miRNAs were predicted.And then,the dynamic"TF-miRNA-lncRNA-gene" immune regulatory networks were constructed,as well as the structural features and regulation mechanism of dynamic regulatory networks were analyzed.Our results might have revealed the structure characteristics of"TF-miRNA-lncRNA-gene" regulatory network and its possible mechanism in the innate immune response of Drosophila.The main results in the study were as follows:1.The expression of miRNAs,lncRNAs and genes showed extensive dynamic changes while Drosophila melanogaster was stimulated by M.luteus.The number of differential expression genes and lncRNAs peaked at 12 h,whereas these differential expression miRNAs were most abundant at 24 h,which showed that the dynamic expression of miRNA and lncRNA could play very important roles in innate immune response of Drosophila.2.The pathway and GO enrichment analysis indicated that differential expression genes are involved in diverse signaling pathways,including Toll and Imd,as well as other signaling pathways,which demonstrated that the cooperation of different signaling pathways plays an important role in the activation of immune response and maintaining homeostasis in Drosophila.In addition,a large number of immune-related genes were up-regulated at 3,12 h and down-regulated at 24 h,suggesting that some genes should be rapidly transcribed at early stage(3,12 h),while others needed to be suppressed.On the other hand,a lot of genes needed to be down-regulated at later stage of the immune response(24 h)to avoid overreaction of the immune system.3.The mutual information expression association network of lncRNA/miRNA showed that non-differential expression lncRNA/miRNA could associate with differential expression lncRNA/miRNA to form some specific correlation clusters of expression,and the clusters did be enriched into different biological pathway to perform specific function.4.The dynamic "TF-miRNA-lncRNA-gene" regulatory network in Drosophila immune response was constructed.The biological analysis of network showed the cross-talk between different signaling pathways in the immune response of Drosophila.Both lncRNAs and miRNAs could coordinately regulate gene expression to activate Drosophila immune response and maintain homeostasis.In addition,a number of new genes and non-coding RNA regulatory factors were identified based on the dynamic "TF-miRNA-lncRNA-gene" regulatory network.5.The biological functions and motifs of the "TF-miRNA-lncRNA-gene"dynamic regulatory network were analyzed,which revealed that miRNAs could regulate the gene expression of different signaling pathways to promote/inhibit the innate immune response in Drosophila.The co-regulation of miRNA and TF could continuely regulate the immune response in Drosophila.It was also revealed that the co-regulation of miRNA and TF motifs always played very important roles in immune response of Drosophila,while co-regulation of IncRNrA and TF motifs were more likely to be involved in activating gene expression in the early stage of immune response in Drosophila.In conclusion,the study revealed that TF,miRNA,IncRNA and gene could coordinate with each other to formed a dynamic regulatory network,which participated in the maintenance of Drosophila immune response and immune homeostasis,revealing that miRNA is key regulation factor,while lncRNA regulates the innate immunity in Drosophila through directly affecting immune-responsive genes at early stage(3,12 h),and lncRNA is more prone to activate immune responses.In particular,non-coding RNA(IncRNA,miRNA)could widely synergistical action with TF to form the dynamic "TF-miRNA-lncRNA-gene"regulatory network to regulate the immune response in Drosophila.These results haverevealed the complex regulatory mechanisms of innate immune responses andhomeostasis maintenance in Drosophila.The present paper not only reveals the complex mechanism of maintaining immune homeostasis in Drosophila,but also provids an important inspiration for further studying the regulation mechanism of immune response in mammalian.