| Grass carp(Ctenopharyngodon idella)is the main farmed fish in my country.The grass carp septicemia induced by Aeromonas hydrophila infection has caused huge economic losses to the grass carp industry.Sepsis is often accompanied by a marked inflammatory response that can lead to septic shock or death if the invading bacteria are not rapidly controlled.A large number of studies have shown that miRNAs are involved in the process of antibacterial immune responses,and many miRNAs in grass carp are involved in inflammatory responses.In animal genomes,miRNAs mainly exist in the form of clusters,and miRNAs in the same miRNA cluster usually regulate functionally related genes.In this paper,the sequence characteristics of grass carp miR-462-731 cluster,the negative feedback loop of ELK1/miR-462-731 and the mechanism of miR-731 targeting ezrin to regulate macrophage phagocytosis were analyzed.The sequence characteristics of the CLDN gene family were also analyzed.,expression and regulatory network were analyzed.The results of this study are as follows:1.Sequence analysis of miR-462-731 clusterIn this study,the precursor and mature sequences of teleost miR-462-731 and mammalian miR-191-425 were obtained from the miRBase database,and multiple sequence alignment found the mature seed sequences of miR-462-731 and miR-191-425.Highly conservative.The phylogenetic tree was constructed and it was found that teleost miR-462-731 clustered together and mammalian miR-191-425 clustered together.After secondary structure prediction analysis,the secondary structure stem-loop structure of teleost miR-462-731 and miR-191-425 was different.2.Double negative feedback loop of ELK1/miR-462-731Alibaba 2.1 predicted that the transcription factor ELK1 could bind to the miR-462-731 cluster promoter,and the dual-luciferase reporter system confirmed the binding of ELK1 to the miR-462-731 cluster promoter,and it was overexpressed in grass carp CIK cell line ELK1,the expression of miR-462 and miR-731 was found to be repressed.RNAhybrid prediction found that ELK1 has a potential binding site of miR-731.Overexpression of miR-731 in CIK cell lines significantly inhibited the expression of ELK1,and inhibition of miR-731 could significantly increase the expression level of ELK1.The results of the dual-luciferase reporter system further demonstrated that ELK1 is a target gene of miR-731.The above data show that miR-731 and ELK1 have a negative feedback regulatory loop.3.miR-731 regulates inflammation and macrophagesThe expression of miR-731 was highest in the spleen and intestine of healthy grass carp.After A.hydrophila infected grass carp,the expression of miR-731 was disordered,which preliminarily indicated that miR-731 was involved in the immune response of grass carp against bacterial infection.In order to study the specific function of miR-731,RNAhybrid was used to analyze the target genes of grass carp miR-731,and ezrin was analyzed as a potential target gene of miR-731.miR-731 was overexpressed and inhibited in CIK cell lines,and fluorescence quantitative experiments confirmed the targeting effect of miR-731 on ezrin.The dual-luciferase reporter system confirmed that ezrin was the target gene of miR-731.Overexpression of ELK1 or inhibition of miR-731 both promoted the expression levels of downstream pro-inflammatory factors(il-1β and tnf-α).Overexpression of miR-731 can also enhance the phagocytic ability of macrophages.The present results contribute to further understanding of the regulatory effect of miR-731 on macrophage function.4.CLDN gene family analysisCldn-5 is another potential target gene of miR-731.The grass carp CLDNs gene family has 60 members,including 29 tandem repeat genes,distributed on 16 chromosomes.In healthy tissues,CLDNs are highly expressed in embryos and kidneys;the expression of cldn-1,cldn-5a,cldn-8b,cldn-31 a and cldn-7a fluctuates after A.hydrophila infection of grass carp.Using String to conduct protein network interactions,it was found that members of the CLDN gene family can interact with each other,and CLDNs proteins and other membrane proteins can also interact with each other.RNAhybrid prediction found that 60 CLDNs genes were targeted and regulated by 70 miRNAs.The complex protein interaction network and the miRNAs-CLDNs regulatory network indicated that the anti-bacterial infection process of grass carp requires a complex regulatory network to maintain body homeostasis. |
