Molecular Mechanism Of PCNA Regulating MITA-mediated Immune Response In Miiuy Croaker(Miichthys Miiuy)

Miiuy croak(miichthys miiuy)is a kind of fish with high economic value produced in Zhoushan ocean of the East China Sea.Miiuy croaker is a kind of fish with high economic value.In recent years,with the expansion of miiuy croaker breeding scale and the change of breeding environment,various diseases have appeared one after another.The disease seriously affects the large-scale cultivation of miiuy croaker.In order to solve this problem,the research on the prevention and control of miiuy croaker disease needs to be carried out urgently.Through the research on the immune mechanism of miiuy croaker,the ways and methods to enhance the ability of miiuy croaker to resist the invasion of pathogenic microorganisms are excavated,so as to provide practical and theoretical basis for the immune mechanism,and then reduce the economic losses caused by diseases to the aquaculture industry.Innate immunity is the first line of defense for the body to prevent the invasion of foreign pathogenic microorganisms.It can remove the pathogenic microorganisms that invade the body,which is an important defense mechanism commonly existing in the body.MITA(mediator of IRF3 activation)is a key protein in the signal pathway of virus induced type I interferon(IFN-I)expression.As the main regulator of the innate immune system for pathogenic microbial infection,it is not only the receptor of the second messenger c GAMP,but also the connector protein of downstream signal molecules.Overexpressed MITA can activate the NF-κB and IRF3(Interferon regulatory factor 3)pathways of type I interferon,so that the body can start the immune response,but these innate immune responses and connector signaling molecules are strictly regulated by the host to prevent excessive immunity,especially the regulation of MITA.PCNA(proliferating nuclear antigen),which exists in the nucleus of dividing cells,can interact with a variety of transcription factors and proteins involved in signal transduction,which is of great significance for the regulation of MITA.Therefore,this study is based on the mechanism of PCNA regulating MITA,and the conclusions are as follows:1.Through gene analysis,comparing the sequence of PCNA between miiuy croaker and other species.It was found that there was a highly conserved amino acid sequence in Rad1 domain.SMART predicted the Rad1 domain of PCNA gene in these species.The conserved structure of PCNA may be related to their functional conserved structure.The three-dimensional structure maps of PCNA genes of miiuy croaker,human and zebrafish were constructed by SWISS-MODEL Repository software.2.PCNA was transfected into EPC cells with different reporter genes(IFN1 and ISRE)by dual luciferase reporter gene assay,and then EPC cells were treated with poly(I:C).The results showed that PCNA inhibits IFN promoter activity under poly(I:C)stimulation.Subsequent experiments with different concentrations of PCNA showed that PCNA had a dose-dependent effect on IFN promoter activation.EPC cells were treated with different concentrations of poly(I:C).The results showed that the inhibitory effect of PCNA was more obvious with the increase of poly(I:C).These data suggest that IFN promoter activity is inhibited in the presence of PCNA.3.Dual luciferase reporter gene was used to detect the expression of IFN promoter activity during co-transfection of overexpressed PCNA and MITA,and the results showed that PCNA overexpression inhibited MITA-mediated IFN promoter activity.Subsequently,the expression of IFN1 and ISRE reporter genes was detected under the concentration gradient and time gradient conditions of PCNA targeting MITA,and the results showed that PCNA negatively regulated MITA in a dose-dependent and time-dependent manner.These results suggest that PCNA can inhibit MITA-induced IFN expression.4.Based on the conclusion that PCNA inhibits MITA-induced IFN expression,it is inferred that PCNA may inhibit the expression of MITA.MKC cells were transfected with overexpressed PCNA,and the expression level of endogenous MITA protein was detected by western blot.The results showed that in the presence of PCNA,MITA protein level decreased.Then,the expression level of MITA protein was detected by concentration gradient and time gradient.The results showed that PCNA inhibited the protein level of MITA in a dose-dependent and time-dependent manner.The cells were transfected with fluorescent MITA-GFP plasmid,and the fluorescence signal was detected when PCNA was overexpressed.The results showed that the fluorescence signal of MITA-GFP was weakened when PCNA was overexpressed.In conclusion,these data suggest that PCNA specifically promotes the degradation of MITA at the protein level.5.This study also carried out experiments on the pathway of PCNA degradation of MITA.First,protease inhibitors and autophagy inhibitors were added to overexpression of PCNA,and the results showed that the degradation of MITA was significantly inhibited by the addition of autophagy inhibitor 3-MA.Dual luciferase reporter gene assay showed that 3-MA hindered the degradation of MITA by PCNA.In conclusion,the negative regulation of PCNA on MITA works through the autophagy pathway.In conclusion,experimental results indicate that PCNA,as a negative regulatory factor of MITA,participates in regulating the immune response caused by pathogenic microbial infection.This study provides a theoretical basis for further exploring the role of protein regulatory mechanism in the immune response regulatory network of fish pathogen infection.This strict negative regulatory mechanism is crucial for reducing inflammatory responses and avoiding excessive inflammation.These data not only provide information about related immune proteins as negative feedback modulators involved in fish anti-bacterial and anti-viral immune responses,but also lay a foundation for the study of signaling pathways involved in fish innate immune responses.