Molecular Regulation Mechanism Of Long Non-coding RNA LTCONS5539 In Miichthys Miiuy

In the long process of evolution,in order to cope with the pressure of a large number of pathogenic microorganisms in the environment,the organisms have developed a complex and accurate anti-infection mechanism which is immune system.Immune system divides into innate immunity and adaptive immunity.Fish,as the most diverse vertebrate in the world,have evolved both innate and adaptive immunity.However,compared with higher vertebrates,the efficiency of fish adaptive immune response is too low to resist the invasion of organisms effectively.Therefore,the innate immunity of fish plays an extremely important role in defending against invasion of microorganisms.Tumor necrosis factor receptor associated factor 6(TRAF6)is an important member of TRAF family which is considered to be an important intracellular multifunctional signal molecule with unique receptor binding function.TRAF6 participates in multiple signal transduction pathways mediated by interleukin-1(IL-1),clusters of differentiation 40(CD40)or other signaling molecules,and ultimately activates nuclear factor kappa-B(NF-κB),activator protein 1(AP1),protein kinase B(PKB)and so on through a series of signal transduction.In recent years,long non-coding RNA(lncRNA)is considered to be a new regulatory transcript which can regulate gene expression at the transcriptional levels through a variety of mechanisms,and regulate function by interacting with protein,RNA,DNA or their combinations.To better promote the innate immune response of fish,it is of great significance to explore the lncRNA regulatory factors associated with TRAF6.The conclusions of this study are as follows:1.After stimulated by Vibrio anguillarum,LPS,poly(I:C)or SCRV,miiuy croaker(Miichthys miiuy)kidney tissue was dissected at different time points and RNA was extracted.The expression of lncRNA LTCONS5539 was detected by quantitative real-time PCR(qRT-PCR).The results showed that the expression of lncRNA LTCONS5539 increased significantly in miiuy croaker after being stimulated by V.anguillarum,LPS,poly(I:C)and SCRV,and reached the maximum after 24 h of stimulation.These results suggested that lncRNA LTCONS5539 can play a regulatory role in miiuy croaker in response to bacterial and viral infection.2.To further characterize lncRNA LTCONS5539,the coding capacity of lncRNA LTCONS5539 was predicted by coding potential calculator(CPC)program.The results showed that the coding potential of lncRNA LTCONS5539 is very low,and the result of western blot further proved that lncRNA LTCONS5539 don’t have coding ability.By comparing with the genome sequence of miiuy croaker,the result showed that the length of lncRNA LTCONS5539 was 549 base pairs(bp),which is located on chromosome 23 and consists of only one exon and in the middle of gene sema4 gb and gene tanc2.In addition,by analyzing the sequence alignment results of miiuy croaker,large yellow croaker(Larimichttys crocea),spotted maigre(Nibea albiflora),red drum(Sciaenops ocellatus)lncRNA LTCONS5539,it can be found that although lncRNA is less conserved among species,lncRNA LTCONS5539 is highly conserved in these closely related species.3.The results of qRT-PCR revealed that lncRNA LTCONS5539 could enhance the expression of inflammatory cytokines and antiviral genes after cells were stimulated with LPS,poly(I:C)and SCRV.Besides,lncRNA LTCONS5539 could inhibit bacterial infection and virus replication.It further indicates that lncRNA LTCONS5539 is involved in anti-bacterial immune response and anti-viral immune response.4.The result of qRT-PCR showed that lncRNA LTCONS5539 promoted the expression of TRAF6 at the mRNA level.Meanwhile,the expression of TRAF6 increased significantly when the cells were transfected with lncRNA LTCONS5539 plasmid and infected with LPS,poly(I:C)and SCRV.In addition,to further determine the role of lncRNA LTCONS5539 on the expression of TRAF6 at the post-transcriptional level,the expression of exogenous and endogenous TRAF6 in cells transfected of lncRNA LTCONS5539 plasmid was detected by western blot.The results demonstrated that lncRNA LTCONS5539 also promote the expression of TRAF6 at the protein level,and TRAF6 protein levels were increased in a dose-dependent manner when overexpression of lncRNA LTCONS5539.5.This study futher explored the regulation of lncRNA LTCONS5539 on TRAF6 mediated signal pathway.The effect of TRAF6 on the activity of different reporter genes was known through previous study.When the cells were transfected with TRAF6 and lncRNA LTCONS5539 plasmid at the same time,the activity of these reporter genes was further up-regulated.The cell activity,cell proliferation and cell apoptosis of cells after transfection with lncRNA LTCONS5539 plasmid were detected.The results showed that lncRNA LTCONS5539 could promote the proliferation and viability of miiuy croaker cell lines and reduce apoptosisIn conclusion,lncRNA LTCONS5539 plays a key role in the antiviral and antibacterial response of miiuy croaker.This study can not only increase the understanding of the function of lncRNA LTCONS5539,but also provide insights into TRAF6 mediated immune response regulatory network.