Molecular Mechanism Of MicroRNA-2187 Involved In Immune Regulation By Targeting TRAF6 In Miiuy Croaker

Miiuy croaker is one of the important marine economic fishes.Because of its delicious meat,its whole body has high utilization value,and its cultivation can bring huge economic benefits.With the gradual expansion of the scale of miiuy croaker aquaculture,the occurrence of diseases has seriously hindered the development of koala and caused huge economic losses.Therefore,there is an urgent need to study the related immune regulation mechanism of the teleost;to provide a theoretical basis for exploring ways and methods to enhance the ability of the teleost to resist the invasion of pathogenic microorganisms,so as to reduce the economic loss caused by pathogenic microorganisms to the aquaculture industry.Innate immunity is also called natural immunity.It can eliminate antigens that invade the body.Innate immunity is different from acquired immunity.This elimination function is non-selective and is an important defense mechanism that exists in the body.As an important intracellular multifunctional signaling molecule,TRAF6 is one of the important members of the TRAF family.It plays an important signal transduction role in many signal pathways.However,the excessive activation of TRAF6 can cause autoimmune diseases.Therefore,TRAF6 is immune to TRAF6.The role of reaction and mediated signaling pathway has attracted many scholars to carry out research on it.More and more evidences show that miRNAs can act as negative regulators to regulate the body’s innate immune response to prevent excessive immune response.In order to avoid excessive immune response caused by pathogenic microorganism infection,it is of great significance to explore miRNAs regulatory factors related to TRAF6.The conclusions of this study are as follows:1.Through the q RT-PCR experiment,in this study,the expression level of miR-2187 in the liver tissue of the miiuy croaker after stimulation by V.anguillarum or LPS and the expression of miR-2187 in the spleen after stimulation by poly(I:C)or SCRV was detected.The experimental results show that the expression of miR-2187 will change significantly under Gram-negative bacteria and virus infection.In addition,miR-2187 mimics and miR-2187 inhibitors were transfected into miiuy croaker kidney cells(MKC),and then the transfected MKC cells were treated with different antigens.The q RT-PCR test results showed that miR-2187 can inhibit inflammatory factors and the expression of antiviral genes,and miR-2187 inhibitor can enhance the expression of inflammatory factors and antiviral genes.2.In this study,miRanda and Targe Scan software were used to predict the potential target genes of miR-2187 including TRAF6,so as to better explore the role of miR-2187 in the immune response.The dual-luciferase reporter analysis verified that miR-2187 can reduce the fluorescent activity of the TRAF6 3’UTR wild-type reporter plasmid,but miR-2187 cannot reduce the fluorescent activity of the mutant reporter plasmid.In addition,a miR-2187 precursor(pre-miR-2187)plasmid was further constructed.The results of a series of targeting experiments proved that the miR-2187 precursor and miR-2187 mimic had similar trends,and both could inhibit TRAF6 3’UTR wild-type reporter plasmid fluorescent activity.3.Experimental results have proved that miR-2187 targets TRAF6 by binding to TRAF6 3’UTR.Next,we used q RT-PCR and Western blot experiments,and the experimental results proved that miR-2187 inhibited the expression of TRAF6 at the m RNA and protein levels,respectively.4.In this study,we explored the way that miR-2187 participates in resisting pathogenic microorganisms by targeting TRAF6,and by detecting the effect of overexpression of TRAF6 expression plasmid in EPC cells on the activity of different reporter genes under induction of different antigens,the experimental results show that overexpression of TRAF6 expression plasmid and the reporter gene has a significant activation effect and further verified that miR-2187 inhibits the activity of different reporter genes by targeting TRAF6.In summary,miR-2187,as a negative regulator,plays an important regulatory role in the TRAF6-mediated signaling pathway.5.Based on the research on the signal transmission effect of TRAF6 in the Toll-like receptor and RIG-I-like receptor signaling pathways,in this study,si RNA experiments were carried out using miiuy croaker kidney cells and analyzed under the stimulation of bacteria and viruses.The expression patterns of inflammatory factors and antiviral genes in fish TRAF6 and related signal pathways mediated by TRAF6.The results of q RT-PCR experiments showed that endogenous TRAF6 was successfully interfered by si-TRAF6,the expression of TRAF6 at m RNA and protein levels decreased,and the expression of inflammatory factors and antiviral genes related to the TRAF6-mediated signaling pathway was significantly down-regulated.In summary,the experimental results show that miR-2187 as a negative regulator is involved in regulating the immune response caused by bacterial and viral infections.This study provides a theoretical basis for in-depth exploration of the role of miRNAs in the immune response regulatory network of fish pathogen infections.This tight negative regulation mechanism is critical for attenuating inflammatory response and avoiding excessive inflammation.These data not only provide the information on miRNA as a negative feedback regulator involved in the antibacterial and antiviral immune response in fish,but enriched miRNA-mediated networks of host-pathogen interactions.