Study On MiRNA Regulation Of Differentially Expressed Genes From Miiuy Croaker Injected With Vibrio Anguillarum

In the evolutionary process,vertebrates have evolved the immune system against pathogens.The immune system is divided into innate immune system and acquired immune system.The Toll/IL-1 receptor superfamily is composed of the TLR family and the IL-1R family.The members of this new signal receptor family contain the TH domain of the Toll homology domain,which is composed of 10 conserved sequences containing 128 amino acid residues.The Toll/IL-1 receptor superfamily is an important pattern recognition receptor that plays an important role in the innate immunity against pathogens.MicroRNAs(miRNA)have been implicated as negative regulators controlling the diverse of biophysical and biochemical processes at the post-transcriptional level.MiRNA is a class of endogenous non-coding small RNA(sRNA)containing about 22 nucleotides,which can cause gene translation blocking or directly mRNA degradation through incomplete complementation of the 3’untranslated region(3’UTR)of the mRNA.In this study,differentially expressed genes from miiuy croaker were obtained by sequencing of two transcriptomes and the miRNA regulation of Toll/IL-1 receptor superfamily was studied.The conclusions are as follows:1.A total of 51,853,370 raw reads and 51,852,199 raw reads were obtained by sequencing the transcriptome of normal miiuy croaker spleen(Ctrl)and spleen injected with Vibrio anguillarum suspension(Van).After alignment with genome and screening,34,790,143 and 34,526,606 clean reads were obtained for Ctrl and Van,respectively.Then these reads were assembled into 47,971 unigenes.By quantitative expression analysis,Together 2,482 significant differentially expressed genes were obtained,containing 1,313up-regulation gene.Then,the GO function analysis,as well as KEGG pathway enrichment analysis,were conducted for these differentially expressed genes.In basis of GO function and KEGG pathway enrichment analysis,we obtained numbers of up-regulation immune related genes,such as TLR1 and IL-1RI of the Toll/IL-1 receptor family,after V.anguillarum infection.2.In this study,we found that IL-1RI was significantly upregulated in Vibrio anguillarum infected spleen tissue and LPS-infected macrophages,which further confirmedthe role of IL-1RI in immune and inflammatory response.Then,we explored the potential roles of miR-192 in regulating interleukin 1 receptor type I(IL-1RI)involved in immune and inflammatory response in miiuy croakers.By means of promoter analysis,miR-192 was found to be an AP-1 dependent gene.Importantly,the dual luciferase reporter assay presented the regulation between miR-192 and IL-1RI.The result of miiuy croaker miR-192 reduced the wild-type IL-1RI but not the mutant one luciferase levels suggested that mmi-miR-192 modulated IL-1RI expression by directly targeting the 3’UTR of IL-1RI mRNA.3.In this study,we used a combination of bioinformatics and experimental techniques to exhibit that miR-217 was the direct negative regulators of TLR1 in miiuy croaker.Furthermore,dual-luciferase reporter assays showed that miR-217 exhibited a greater negative regulatory effect on TLR1.Additionally,we also demonstrated that the expression of miR-217 could be up-regulated by LPS stimulation in either LPS-stimulation spleen tissue or LPS-treated cultured macrophage,which indicating thatmiR-217 could be induced by LPS and may be as the negative regulators of TLR1 involved in the immune response to LPS stimulation.These results would enhance our understanding of the miRNA regulation in fish TLR signaling pathways.