Molecular Mechanism Of Micro Rnas Regulating IκBα Gene In Miichthys Miiuy

Simple fishing and traditional small-scale farming can no longer meet daily needs.Therefore,the industrialization and scale of aquatic product farming are inevitable with the increasing demand for aquatic products.And as the breeding density increases,once a large-scale infection occurs,it will cause huge economic losses.Fish defense against disease mainly relies on innate immunity.We can explore and enhance the ability of miiuy croaker to resist pathogenic microorganisms,reducing the economic losses caused by pathogenic microorganisms to the aquaculture industry.In teleosts,the innate immune system is the host’s first line of defense against pathogens.It cooperates with the adaptive immune system to help the host survive in hostile environments.Activation of nuclear factor kappa B(NF-κB)signaling pathway is considered to be an important process in the process of inflammation.And IκBα(inhibitorα of NF-κB),it can inhibit the NF-κB signaling pathway and prevent NF-κB from binding to DNA,so it plays a very important role in innate immune regulation.MicroRNAs(miRNAs)is a small noncodingRNA that has been shown to be a negative regulator that regulates many biochemical processes,such as cell growth,proliferation and immune responses.In this study,we took miRNA as the starting point to explore whether miRNA targets IκBα regulates NF-κB,and affects the innate immunity of miiuy croaker.The conclusions of this study are as follows:1.This study found that miR-144 and miR-122 have the same potential target gene IκBα by using miRanda and Target Scan software.In order to verify whether miR-144 and miR-122 target the 3’UTR of IκBα.The dual-luciferase reporter gene plasmid of wildtype IκBα-3’UTR and the dual-luciferase reporter gene plasmid of mutant miR-144 and miR-122 with the 3’UTR complementary site of IκBα were constructed.They cotransfected with miR-144 and miR-122 mimics in epithelioma papulosum cyprini(Epithelioma Papulosum Cyprini)cells.The luciferase activity was detected,and the results indicated that miR-122 and miR-144 target IκBα as negative regulators actor.2.It has been confirmed that miR-122 and miR-144 target IκBα,in order to verify the level of regulation of IκBα gene expression.The mimics of miR-122 and miR-144 were transfected into Miichthys miiuy intestine cells(MIC).The results of western blot showed that miR-122 and miR-144 mimics inhibited the protein level of IκBα.The results of q PCR experiments confirmed that miR-122 and miR-144 The expression of IκBα was inhibited from the mRNA level.At the same time,the inhibitor of miR-122 and miR-144 were transferred into MIC cells,and the results obtained were contrary to the mimics,which promoted the expression of IκBα at the mRNA and protein levels.It was confirmed that miR-122 and miR-144 act as negative regulators to inhibit the expression of IκBαfrom the protein and mRNA levels.3.In this study,it was found that overexpression of p65 can activate the NF-κB,interleukin-8(IL-8)and interleukin-1β(IL-1β)signaling pathways,while the expression of IκBα inhibits this activation.4.Based on the inhibition signaling pathway by IκBα.Luciferase reporter gene plasmid,IκBα plasmid,and p65 plasmid were cotransfected with miR-144 and miR-122 mimics,precursors and inhibitors in this study.The results showed that miR-122 and miR-144 targeted to inhibit IκBα and promote p65-mediated activation of signaling pathways such as NF-κB.Taken together,the experimental results suggest that miR-122 and miR-144 are negative regulators that target the 3’UTR of IκBα and inhibit expression at the mRNA and protein levels,thereby promoting the activation of NF-κB signaling pathway by p65.This study provides a theoretical basis for the mediation of miRNAs in the immune signaling pathway in teleost fish.miRNAs,as a negative regulator,are usually inhibitory to the activation of the pathway,while the miRNAs in this paper target IκBα,and because of the functions possessed by the target genes,miRNAs act as a facilitator of the pathway,promoting the innate immune response of the organism,which may be beneficial to help the host to defend against pathogenic invasion and suggest a new theoretical basis for enhancing immunity.In this paper,miRNAs are used as a link in this paper to enrich the immune regulatory network of non-codingRNAs in teleosts fish.