Study Of MiRNA Modulating NF-κB Signaling In Miiuy Croaker

The innate immune system is the first line of host defense against invading pathogens.The main molecules that identify invading pathogens are pattern recognition receptors(PRRs).There is increasing evidence that the activation and termination of immune responses are regulated by various endogenous regulatory molecules and mechanisms.Micro RNAs(miRNAs)are a group of small non-coding RNAs that regulate gene expression by binding to the 3 ’untranslated region(UTR)of the target gene,which ultimately leads to blocked protein translation or m RNA degradation.It has been found that miRNAs are involved in the regulation of various biological processes,including proliferation and differentiation,cancer,and apoptosis.Up to 60% of m RNAs in mammals are regulated by miRNAs.Toll-like receptor-mediated NF-κB signaling pathway is a key signaling pathway that regulates the innate immune response.TLR is a pattern recognition receptor mainly located on the cell surface.It can mediate the immune response to various specific pathogens by activating the transcription factor NF-κB signaling pathway.Recent studies have found that miRNAs are also a key component of complex regulatory networks in fish immune responses.In this study,different levels of signaling molecules in TLR-mediated NF-κB signaling pathway were selected as targets,with the aim of discovering miRNAs involved in regulating these key signaling molecules.This will provide theoretical basis for the future functional research and development of fish miRNAs as disease diagnosis and treatment.The specific research content is divided into the following aspects:1.Screening of miRNAs regulating My D88 and study of its mechanismWith the exception of TLR3,all mammalian TLRs use My D88 to transmit signals.As an important singnaling molecule,miRNAs seem to regulate the singnaling molecules more effectively than directly targeting TLRs.This study uses bioinformatics methods to predict miRNAs that may bind to the 3’UTR region of My D88.Then,several miRNAs were screened,including miR-3570,miR-214,and miR-148 by using the dual-luciferase reporter assays,which could negatively regulate the luciferase activity.Using real-time quantitative PCR and western blotting,it was confirmed that miR-3570 and miR-214 can reduce m RNA stability and negatively regulate the protein expression of My D88.miR-148 has been confirmed to negatively regulate the expression of My D88 from the protein level,and the negative regulation is achieved by inhibiting the translation of the protein rather than affecting m RNA stability.Subsequently,the role of these three miRNAs in the LPS-induced inflammatory response was confirmed,and they were found to regulate the inflammatory response by inhibiting the expression of inflammatory cytokines.Through the dual-luciferase reporter assays and knockdown of My D88 gene experiments,it was confirmed that miR-3570,miR-214,and miR-148 can regulate the My D88-mediated NF-κB signaling pathway.2.Screening of miRNAs regulating IRAK4 and its mechanismIRAK4 is known as a key adaptor kinase and has proven to be an important molecule in TLRdependent immune responses.This study uses bioinformatics methods to predict miRNAs that may bind to the IRAK4 3’UTR region.Then using the dual-luciferase reporter assays,two miRNAs,including miR-203 and miR-21,were screened to negatively regulate luciferase activity.Using western blotting and real-time quantitative PCR experiments,it was confirmed that miR-203 negatively regulates IRAK4 protein expression by reducing m RNA stability.miR-21 has been confirmed to negatively regulate IRAK4 expression from the protein level,and the regulation is achieved by inhibiting the translation of the protein rather than affecting its m RNA stability.It was subsequently confirmed that miR-203 and miR-21 can negatively regulate the expression of inflammatory cytokine induced by LPS.Further experiments confirmed that miR-203 and miR-21 can regulate the IRAK4-mediated NF-κB signaling pathway under LPS stimulation.The above experimental results that miR-203 and miR-21 regulates the expression of IRAK4 gene have also been found in other teleost fish.3.Screening of miRNAs regulating p65 and its mechanismsActivation of the NF-κB pathway results in the transcription of a variety of genes,such as cytokines,chemokines and adhesion molecules.Most bacteria can bind to receptors on the surface of cell membranes,thereby triggering the NF-κB signaling pathway to change gene expression to regulate the host immune responses.This study uses bioinformatics methods to predict miRNAs that may bind to the p65 3’UTR region.Then using the dual-luciferase reporter assays,we screened out that miR-216 a can negatively regulate luciferase activity.Using real-time quantitative PCR and western blotting technology,it was confirmed that miR-216 a can reduce m RNA stability and negatively regulate p65 protein expression.By studying the transcriptional regulation of miR-216 a,it was proved that two Ap1 binding sites and four Sp1 binding sites in the miR-216 a promoter are essential for the transcription of miR-216 a.It was subsequently confirmed that miR-216 a plays a negative regulatory role in the LPS-induced inflammatory response.Through the dual-luciferase reporter assays,it was confirmed that miR-216 a inhibits the expression of inflammatory factors by regulating the NF-κB signaling pathway.We also verified that the above experimental results that miR-216 a can target the expression of p65 gene,also found in other telesot fish.