Identification And Functional Study Of Circular RNA CircRara In Miichthys Miiuy

The immune system is responsible for monitoring and defending the invasion of different exogenous pathogens,and maintaining internal homeostasis by maintaining appropriate immune tolerance and regulation.At the beginning of the organism’s infection with pathogens,innate pattern recognition receptors(PRRs)participate in mediating to resist the invasion of pathogens.It includes Toll-like receptor,NOD-like receptor and C-type lectin receptor.These receptors can detect the presence of pathogenic microorganisms,such as sensing pathogen-related molecular patterns(PAMP)and damage-related molecular patterns(DAMP).After identifying these invasive pathogens,they will initiate corresponding mechanisms to eliminate these potential infection threats to the body.Among them,PAMP is a conservative structure between microbial species,and DAMP is an endogenous molecule released by damaged cells.The intracellular signal triggered by several PRRs receptors triggers the intracellular signal cascade,leading to the transcription and expression of inflammatory mediators,activating the humoral and cellular mechanisms for eliminating pathogens,and coordinating the clearance of pathogens and infected cells.In recent years,circRNA has been proved to be widely involved in a variety of biological processes,which can affect the transcription silencing,translation or decay of m RNA.In addition,circRNA are related to the development of tissues and organs,and may play a role in various diseases.Exon circular RNA(circRNA)is a covalently closed RNA molecule produced during gene reverse splicing.Some studies have reported that some circRNA interact with micro RNAs,some have been translated,and circRNA have been shown to regulate immune response and behavior.In order to further study the effect of circRNA on the innate immune response of teleost fish,we explored the regulatory role of circRara in the innate immune response of Miichthys miiuy.The research is as follows:1.Through qRT-PCR,it was found that the expression of circRara increased remarkably after LPS or poly(I:C)stimulation,and then further analysis was carried out by qRT-PCR.The research showed that the expression of circRara in the spleen of M.miiuy was remarkably increased after LPS or poly(I:C)stimulation,which indicated that circRara might regulate the immune response of M.miiuy.These results indicate that circRara plays a regulatory role in M.miiuy,thereby affecting the stimulation of bacteria and viruses.2.In order to further describe the characteristics of circRara,we first predict it through bioinformatics.The results showed that circRara is located on chromosome 23 of M.miiuy,consisting only of the starting and ending sequences of the fourth exon through splicing.The specific size of this circRara after reverse splicing is 796 bp.To determine whether head and tail splicing is a result of trans splicing or genome rearrangement,we subsequently used several universal circRNA detection methods.We designed different primers to amplify circRara.The results of Sanger sequencing confirmed that circRara is a head and tail splice in qRT-PCR products.In addition,since stability is considered one of the most important characteristics of circRNA,we amplified circRara from c DNA using primers,but no amplification products were observed from g DNA.RNase R was used to confirm the cyclization of circRara.Therefore,it has been proven that circRara and nonlinear Rara or glyceraldehyde-3-phosphate dehydrogenase(GAPDH)can resist the digestion of RNase R.3.Through the qRT-PCR experiment,it was found that the successful construction of the circRara overexpression plasmid increased the expression level of circRara m RNA in MSp C cells,rather than the expression level of linear Rara m RNA,which further verified the regulatory role of circRara.After that,the results showed that the expression of inflammatory cytokines and antiviral genes increased after the cells received LPS and poly(I:C)stimulation.In addition,after receiving LPS and poly(I:C)stimulation,the expression of anti-inflammatory factors and antiviral genes in MSp C cells gradually increased with the increase of the concentration of circRara.It further showed that circRara was involved in the anti-bacterial immune response and anti-viral immune response.4.In order to further verify the reliability of this research,we studied the regulatory effect of circRara on cytokines at the luciferase level.First,we tested whether circRara regulates the activation of the NF-κB and IRF3 signaling pathways.We transfected the circRara plasmid into EPC cells,and the results showed that circRara increased luciferase activity in a time-dependent manner.After that,we also detected the activity and proliferation of the cells after transfection of the circRara plasmid.The results showed that circRara could promote the proliferation and vitality of the spleen cells of M.miiuy.To sum up,it is proved that circRara plays a role in the antiviral and antibacterial reactions of M.miiuy and enhances the innate immune response of M.miiuy by double luciferase activity detection,qRT-PCR detection,cell proliferation,activity and other experiments.