Two Noncoding RNAs Modulate Innate Immune Responses In Miiuy Croaker

The innate immune system is the host’s first line of defense against pathogen infection.The host can recognize pathogen associated molecular patterns(PAMPs)through pattern recognition receptors(PRRs)and trigger a series of innate immune responses.Viral nucleic acid is a typical PAMP,which can induce innate immune response after being recognized by PRRs.In host cells,there are two main types of PRRs,which play a crucial role in the recognition of viral nucleic acids,namely Toll-like receptors(TLRs)and RIGI-like receptors(RLRs).Unlike TLRs,RLRs is a key sensor of viral RNA in host cytoplasm,which can recognize multiple viral RNA to trigger antiviral innate immune response.When RNA virus infects host cells,RLRs family members RIG-I and MDA5 interact with mitochondrial antiviral signaling(MAVS)proteins,which subsequently transmit signals through TANK-binding kinase 1(TBK1)and canonical IKK complex(α/β/γ)to transmit signals to activate interferon regulatory factor 3(IRF3)and nuclear factor κ B(nuclear factor kappa-B,NF-κB)signal pathway.The mediator of IRF3 activation(MITA)is a mitochondrial and ER-associated membrane protein,which has been shown to mediate the production of interferon(IFN)and inflammatory factors,participate in the innate antiviral response,and respond to RNA and DNA viral infections.It has been confirmed in RLRs-mediated transmission of innate immune signals plays a vital role.After RLRs recognize viral RNA,MITA responds and activates a series of signaling pathways,which in turn triggers the innate antiviral response.When viral RNA appears in the cytoplasm,MITA is stimulated and activated by signaling molecules from upstream such as MAVS,and then recruits and activates TBK1 and IKKβ,which in turn activates IRF3 and NF-κB signaling pathways.In previous studies,we found that the expression of MITA in miiuy croaker(Miichthys miiuy)was significantly up-regulated under viral infection,indicating that MITA has obvious antiviral effect.In view of the indispensable role of MITA in the antiviral immune response,so MITA was selected to further study its signal transduction mechanism.As a highly conserved noncoding RNA(nc RNA),micro RNA(miRNA)has only 21 to 24 nucleotides in length.Mi RNA is an important post transcriptional regulator of gene expression,which plays a role by directly pairing with the target site in the 3’untranslated region(3’UTR)of m RNA.In addition,studies have shown that miRNA can regulate gene expression and participate in a variety of physiological and pathological development processes,such as regulating cell proliferation and inducing cell apoptosis.Many studies have proved that many coding genes modify the antiviral signal transduction mediated by MITA,but the regulatory effects mediated by noncoding genes are still lacking.More and more evidences show that miRNA plays a key role in the regulation of viral infection.However,at present,miRNA is mainly studied in plants and mammals,while the research of this kind in fish is still relatively lacking.As a kind of nc RNA with covalent closed loop structure,the significance of circular RNA(circ RNA)is gradually being explored.More and more evidence showed that circ RNA can be used as a miRNA sponge to control gene transcription.In addition to being a miRNA sponge,circ RNA has a variety of regulatory mechanisms in cell processes,including selective splicing and regulation of gene expression,regulation of r RNA and biogenesis of t RNA.Interestingly,some research groups reported the latest progress of circ RNA and revealed its new functions.For example,the recognition of circ RNA by receptor RIG-I is crucial for the transmission of innate immune signals,indicating that circ RNA has the function of immune regulation.In conclusion,these findings demonstrated the powerful biological function of circ RNA,which can play an important role in immune activation and virus or bacterial infection.Therefore,we conducted the following research.1.The expression of miR-27 c was up-regulated in the spleen tissue of miiuy croaker treated by poly(I:C)by quantitative real-time PCR(q RT-PCR)assay.It suggests that miR-27 c may participate in immune response.In addition,we analyzed the expression of circ RNA in the spleen tissues of the treated and untreated groups through RNA sequencing data,and found that after treatment with SCRV and LPS,circ Plce1 was significantly up-regulated.Then,through further analysis by q RT-PCR,the expression of circ Plce1 in the spleen tissues of miiuy croaker stimulated by poly(I:C)and LPS was significantly increased,indicating that circ Plce1 might participate in the immune response of miiuy croaker.2.To further explore the biological functions of nc RNA miR-27 c and circ Plce1,miR-27 c and the constructed circ Plce1 overexpression plasmid were transfected into miiuy croaker intestinal cells respectively.The results of q RT-PCR showed that miR-27 c could negatively regulate the expression of antiviral factors,while overexpression of circ Plce1 could significantly promote the expression of antiviral factors and inflammatory factors.This indicates that miR-27 c and circ Plce1 are involved in the innate immune regulation of miiuy croaker.3.Use the miRanda and Targetscan program to predict the target gene of miR-27 c,and predicted that miR-27 c may target MITA.In order to confirm the reliability of this prediction result,we used dual luciferase reporter gene assay and green fluorescent protein(GFP)expression experiment to verify the regulatory effect of miR-27 c on MITA.The results showed that miR-27 c could down-regulate the luciferase activity of wild type MITA 3’UTR,but had no effect on the luciferase activity of mutant type MITA 3’UTR.The activity of green fluorescence detected by a microplate reader also confirmed the targeting effect of miR-27 c on MITA.In addition,the pre-miR-27 c plasmid was constructed,and the dual luciferase reporter gene experiment and GFP expression experiment were also carried out,which confirmed that the pre-miR-27 c can also negatively regulate the MITA 3’UTR.Similarly,the dual luciferase reporter gene experiment was performed on circ Plce1,and it was found that it can activate NF-κB and IRF3 signaling pathways up-regulate the luciferase activity of the reporter gene.In conclusion,these results fully proved that MITA is the target of miR-27 c,and circ Plce1 participates in the antiviral and anti-bacterial immune response of miiuy croaker.4.Through western blot and q RT-PCR experiments,it was proved that miR-27 c could target the 3’UTR region binding to MITA and reduced the expression of MITA at the m RNA and protein levels.Through cell proliferation and cell activity experiments,it was proved that overexpression of circ Plce1 can promote cell proliferation and increase cell activity,thus participating in innate immune response.5.By poly(I:C)stimulation,it was further proved that miR-27 c targeted MITA and participated in the antiviral immune response of miiuy croaker.However,circ Plce1,stimulated by poly(I:C)and LPS,can significantly promote the expression of antiviral genes and inflammatory factors,thus enhancing the innate immune response of miiuy croaker.