Study Of The Regulatory Mechanisms Of Japanese Flouder MicroRNA Pol-miR-194a And Pol-miR-3p-2 And Investigation Of Their Roles In Edwardsiella Tarda Infection

As an important mechanism of post-transcriptional modulation,microRNA(miRNA)participates in the regulation of multi-biological processes.During the battle between pathogen invasion and host defense,miRNAs also play a crucial role.A number of Japanese flounder miRNAs have been discovered in previous studies in our laboratory.In this thesis,two flounder miRNAs identified before,i.e.pol-miR-194 a and pol-miR-3p-2,were selected for the study of their regulation mechanisms and involvement in the infection of Edwardsiella tarda.Firstly,we examined the response of pol-miR-194 a expression during E.tarda infection.By quantitative real time PCR(qRT-PCR)method,we discovered that the expression level of pol-miR-194 a in liver,spleen and gill of flounder infected by E.tarda was significantly up-regulated at 24 hours post infection.Luciferase report assay revealed interaction between pol-miR-194 a and the 3'UTR of interferon regulatory factor 7(IRF7),indicating that IRF7 is a target gene of pol-miR-194 a.Mutation analysis showed that the interaction between pol-miR-194 a and the 3'UTR of IRF7 relied on the specific seed sequence of pol-miR-194 a.Western blot analysis showed that over-expression of pol-miR-194 a in flounder FG-9307 cells significantly decreased the protein level of endogenous IRF7.When pol-miR-194 a mimic and the promoter reporter plasmid of type I interferon(IFN)were co-transfected into FG-9307 cells,the promoter activity of type I IFN was markedly inhibited.By in-vitro over-expression and intracellular replication assay,we discovered that pol-miR-194 a could enhance the intracellular replication of E.tarda.The above results indicated that the expression of pol-miR-194 a was regulated by E.tarda;the up-regulated polmiR-194 a could then inhibit the expression of IRF7 and type I interferon,leading to an attenuated immune response,which finally enhanced the intracellular replication of E.tarda.Autophagy plays an important role in many biological processes including growth and development,tumorigenesis,inflammatory response,pathogen infection and host immune response.In this study,we found that the infection of E.tarda induced autophagy in the spleen cells of Japanese flounder and in FG-9307 cells,a cell line of Japanese flounder.p53,which plays an important role in autophagy process,was predicted to be a target gene of a flounder miRNA named pol-miR-3p-2.By qRT-PCR method,we found that in flounder infected with E.tarda,the expression levels of pol-miR-3p-2 in the liver,spleen and gill of the fish were significantly upregulated at 24 hours post infection.Through luciferase reporter assay and western blot analysis,p53 was confirmed to be a target of pol-miR-3p-2.When overexpressed in FG-9307 cells,pol-miR-3-2 could induce the autophagy process.Further experiment discovered that,when p53 was knocked-down in FG-9307 cells,the autophagy related gene of beclin was significantly up-regulated.Similar results were obtained in the experiment with HEK293 T cells.By in vitro over-expression and intracellular replication assays,we found that pol-miR-3p-2 could inhibit the intracellular replication of E.tarda.These results indicated that,infection of E.tarda promoted the expression of pol-miR-3p-2,which decreased p53 protein level and enhanced the expression of beclin,which finally promoted autophagy in Japanese flounder and inhibited the intracellular replication of E.tarda.In summary,we have elucidated the regulation mechanisms of two flounder microRNAs,pol-miR-194 a and pol-miR-3p-2,and revealed their effects on E.tarda infection.The results of this study add new insights into the function of fish miRNAs and the infection mechanism of E.tarda.