Identification And Regulation Of Two Immune Related MicroRNA Target Genes In Miiuy Croaker

In the long evolutionary process of organisms,vertebrates have evolved a better immune system toagainst external pathogens infection.The vertebrate immune system,which is an important part of their defense against diseases,it’s can be divided into Innate Immunity System and Adaptive or Acquired Immunity System.Toll-like receptors(TLRs)plays a key role in the organism’s identification of pathogens.MicroRNAs(miRNAs)are an abundant class of small RNA molecules with endogenous and noncoding features of approximately 18-25 nucleotides,which can induce target gene mRNA degradation or inhibit translationat the post-transcriptional level and regulates various physiological processes.When the body is infected with thepathogens,the immune cells recognize the components of the pathogens specific by pattern recognition receptor and activate downstream signaling pathways,then cause the production of pro-inflammatory cytokines and type I interferons.In this study,we analyzed the transcriptome data of miRNA by high-throughput sequencing that existed in the laboratory before and after infection with Vibrio anguillarum,and screened differentially expressed miRNAs related to pathogen infections,and several of these miRNA were studied for the target regulation of the Toll-like receptors,and the conclusions are as follows:1.By analyzing the transcriptome data of miRNAs before and after infection of Vibrio anguillarum by high-throughput sequencing,and the differentially expressed miRNA to predicted for the target genes that could be regulated,including the Toll receptor family of TLR14 and TLR13.2.In this study,by miRanda and TargetScan software,we predicted that miR-122 may target to TLR14.Then,we found the expression of TLR14 was significantly increased in spleen tissue infected with Vibrio anguillarum and the macrophages of stimulated by LPS,while the expression of miR-122 was decreased in the corresponding time points as to the above in spleen tissue and the macrophages.Real-time quantitative showed that mi R-122 may be targeted to TLR14.By dual luciferase reporter assays,the results show that miR-122 inhibited the wild-type of TLR14 3’UTR reporter plasmid fluorescence activity,but not the similar inhibition of the mutant further confirm the role of miR-122 inthe targeted regulation of TLR14.3.TLR13 is involved in immune response and inflammatory reactions in the body.In this study,we found that the expression levels of TLR13 were significantly increased in the spleen tissue of miiuy croaker and the LPS-stimulated macrophages.TargetScan,PITA,mi Randa,and other miRNA target gene prediction software predict potential miRNAs that might target the TLR13 3’UTR.Then,by dual luciferase assays,mi R-8159 has the strongest inhibitory effect on the activity of TLR13 3’UTR reporter plasmids.Then,after further double luciferase assays,it was concluded that miR-8159 not only significantly inhibited the activity of the luciferase of the wild-type reporter plasmid TLR13 3’UTR,but also had no inhibitory effect on the mutant type reporter plasmid.It was confirmed that the mi R-8159 mimic can directly target the "seed region" of the TLR13 3’UTR.In addition,a series of targeted genes experiments were carried out by using cloning vectors constructed mi R-8159 precursors plasmid,the result showed that the pre-miR-8159 plasmid have inhibitory effects similar to mi R-8159 mimics.The pre-miR-8159 plasmid target acts on the activity of the wild-type reporter plasmid TLR13 3’UTR,whereas there is no similar inhibitory effect on the mutant reporter plasmid.We also found that the overexpression of mi R-8159 negatively regulates the expression of TLR13 in macrophages,however,the inhibitory effect of miR-8159 on target gene was decreased after transfection with mi R-8159 inhibitors.In addition,through the results of real-time fluorescence quantification showed that the expression of miR-8159 was significantly increased in macrophages stimulated by LPS and in spleen tissue of miiuy croaker infected with Vibrio anguillarum.All experimental results indicate that miR-8159 may be involved in the body’s response to the regulation of Vibrio anguillarum infection.