Molecular Regulation Mechanism Of Long Noncoding RNA LTCONS6801 In Miichthys Miiuy

The development of sequencing technology has promoted the study of non-coding genes,which play a regulatory role on coding genes to influence the final gene expression.Non-coding genes with long sequence length(>200nt)are called long non-coding genes(lncRNA),and many members of non-coding genes are classified into lncRNA.LncRNA can interact with genes and proteins,so they can be involved in related pathways to regulate various physiological and pathological processes,including immune pathways.Starting from gnathic vertebrates,immunity is divided into innate immunity and adaptive immunity.The synergistic effect of innate immunity and adaptive immunity provides a strong defense line for the host to resist the invasion of foreign pathogens.Innate immunity,the host’s first line of defense,initiates the expression of downstream cytokines or antiviral factor through nonspecific recognition of pathogen-associated molecular patterns(PAMPs).Host pattern recognition receptors(PRRs)are primarily responsible for recognizing PAMPs during pathogen invasion.After recognizing PAMPs,PRRs send signals to downstream pathways to activate cytokine or interferon release,further activating host-specific immune responses.In response to a series of conformational changes and kinases,the immune signaling pathway initiates the type I interferon(IFN-1)pathway to activate immune-related genes and inflammatory factors.Interferon alters the expression of hundreds of interferon-stimulating genes(ISG),leading to changes in the cellular transcriptome and the release of antiviral factors,including Mx1 and Viperin.Miiuy croaker(Miichthys miiuy)is a kind of marine fish with both economic and scientific value.It is an excellent model to study the mechanism of innate immune regulation.The aquatic environment in which fish live provides conditions for the spread of pathogens,which is easy to cause a large number of infection and even death of the breeding population in a short time.The study of immune related pathways in fish provides feasible strategies to promote innate immunity in fish at the molecular level,while enriching innate immune signaling pathways.TANK-binding kinase 1(TBK1)is a kinase that is also present in the TLR3 and RLRs signaling pathways,mediates the interferon pathway and thus exerts antiviral functions.Some studies have shown that TBK1 selectively up-regulates or inhibits downstream pathway members such as interferon regulatory factor 3(IRF3),IFN-1 and Mx1 to avoid immune inflammation caused by excessive positive feedback on antiviral function of TBK1.The conclusions of this study are as follows:1.LncRNA LTCONS6801 is a non-coding RNA with a length of 674 nucleotides located on chromosome 7 of miiuy croaker,and the full length sequence of lncRNA LTCONS6801 in miiuy croaker was obtained.The homologous sequence alignment of the lncRNA LTCONS6801 of three teleosts fishes,red drum(Sciaenops ocellatus),spotted maigre(Nibea albiflora)and miiuy croaker,showed that the lncRNA LTCONS6801 was highly conservative among the species with close blood relationship.2.The results of cell viability and cell proliferation experiments showed that lncRNA LTCONS6801 significantly increased the viability of miiuy croaker cells.3.Overexpression of lncRNA LTCONS6801 increased the expression level of TBK1.Further research was conducted,quantitative real-time PCR(q RT-PCR)experiments found that overexpression of lncRNA LTCONS6801 increased the m RNA expression level of TBK1.Western blot experiments showed that lncRNA LTCONS6801 up-regulated the expression of TBK1 and endogenous TBK1 in a dose dependent manner.This indicates that lncRNA LTCONS6801 up-regulates the expression of TBK1 at both the transcriptional and post transcriptional levels.Then,the regulatory effect of lncRNA LTCONS6801 on TBK1 pathway was studied.Due to TBK1 activation of IRF3 and nuclear factor kappa-B(NF-κB)to mediate the interferon pathway,lncRNA LTCONS6801 can significantly increase the expression levels of IRF3,NF-k B,and IFN-1 reporter genes in the presence of TBK1,and exhibit time and dose dependent effects.Indicating that lncRNA LTCONS6801 can promotes TBK1 mediated immune signaling pathway.4.After poly(I: C)stimulation and stimulation of Siniperca chuatsi rhabdovirus(SCRV)infection,lncRNA LTCONS6801 significantly increased the m RNA expression level of antiviral genes(Viperin,IFN-I,and Mx1).It indicates that lncRNA LTCONS6801 participates in the regulation of antiviral pathway.5.After poly(I:C)stimulation and SCRV infection,lncRNA LTCONS6801 also up-regulated TBK1 expression.Studies on IRF3,NF-k B,and IFN-1 reporter genes show that lncRNA LTCONS6801 promotes expression of TBK1 mediated signaling pathway molecules after poly(I:C)stimulation and SCRV infection.This indicates that lncRNA LTCONS6801 promotes the TBK1 mediated antiviral pathway.To sum up,it is concluded that lncRNA LTCON6801 regulates TBK1 mediated innate immune antiviral signaling pathway in miiuy croaker,and as a positive regulator in the innate immune pathway.This study provides a theoretical basis for lncRNA to regulate the antiviral immunity of teleost TBK1,and also provides a reference for the importance of lncRNA in the innate immune response of lower vertebrates.