Study On The Function Of Edwardsiella Tarda-induced MicroRNAs Of Paralichthys Olivaceus

Japanese flounder(Paralichthys olivaceus)is an important aquaculture fish species farmed in China.It is susceptible to infection by several fish pathogen,including Edwardsiella tarda.E.tarda is a Gram-negative bacterium with a wide host range.MicroRNAs(miRNAs)are a type of single-stranded,non-coding RNAs consisting of 21-23 nucleotides and participate in the regulation of multi-biological processes,including pathogen infection,cellular autophagy,and cellular apoptosis,by negatively regulating the expression of their target genes at the post-transcriptional level.In this dissertation study,we employed high-throughput sequencing technology to examine miRNAs expression in the kidney of Japanese flounder infected with E.tarda at different time points.A total of 96 miRNAs showed differential expression(named DEmiRNAs)after E.tarda infection.The target gene prediction analysis was carried out for these 96 DEmiRNAs,and a total of 2779 potential target genes were found.Enrichment analysis of target genes revealed that these 2779 potential target genes were associated with diverse biological processes,cellular components,and molecular functions,and regulated diverse pathways related to immunity.Among the above 96 DEmiRNAs,four DEmiRNAs with large fold change or abundant expression,i.e.pol-miR-182-5p,pol-miR-novel＿171,pol-miR-novel＿547 and pol-miR-novel＿395,were selected for the study of regulatory mechanisms during the infection of E.tarda.1.For pol-miR-182-5p,we found that the target gene of pol-miR-182-5p was sphingosine-1-phosphate receptor 1(S1PR1).pol-miR-182-5p regulated Japanese flounder S1PR1(PoS1PR1)negatively via the specific interaction with the 3′UTR of PoS1PR1.Overexpression of pol-miR-182-5p in flounder cells promoted apoptosis and inhibited cellular proliferation.Knockdown of PoS1PR1 in flounder enhanced E.tarda invasion and dissemination in fish tissues.2.For pol-miR-novel＿171,we found that pol-miR-novel＿171 expression was regulated by E.tarda and megalocytivirus(RBIV-C1)infection.Family with sequence similarity 49 member B(FAM49B)was the target gene of pol-miR-novel＿171,and was negatively regulated by the latter.Japanese flounder FAM49B(PoFAM49B)expressed in multiple tissues of flounder,and recombinant PoFAM49B(r PoFAM49B)interacted with Gram-negative bacteria and inhibited the growth of E.tarda and Pseudomonas fluorescens.Interference with PoFAM49 B expression in flounder cells promoted the intracellular replication of E.tarda.Similar effects on the intracellular replication of E.tarda were observed with pol-miR-novel＿171 overexpression.Consistently,in vivo knockdown of PoFAM49 B in flounder enhanced E.tarda dissemination in fish tissues.Furthermore,interference with PoFAM49 B expression,or overexpression of pol-miR-novel＿171 in flounder cells,promoted cellular apoptosis,while in vitro and in vivo knockdown of PoFAM49 B augmented the expressions of pro-apoptotic genes.3.For pol-miR-novel＿547,we found that the target gene of pol-miR-novel＿547 is phosphatase and tensin homolog(PTEN).It negatively regulated the expression of Japanese flounder phosphatase and tensin homolog(PoPTEN)in m RNA and protein levels.E.tarda and RBIV-C1 could affect the expression of pol-miR-novel＿547 and PoPTEN.Interference with PoPTEN expression or overexpression of pol-miR-novel＿547 in flounder cells promoted E.tarda invasion.Consistently,in vivo knockdown of PoPTEN enhanced E.tarda dissemination in flounder tissues,whereas in vivo overexpression of PoPTEN attenuated E.tarda dissemination but facilitated RBIV-C1 replication.Further studies showed that PoPTEN affected autophagy activation via the AKT/m TOR pathway and also modulated the process of apoptosis.4.For pol-miR-novel＿395,we found that it targeted the gene of poly(U)-binding-splicing factor 60 k Da(PUF60).The expression of pol-miR-novel＿395 and Japanese flounder PUF60(PoPUF60)was regulated by E.tarda and RBIV-C1,and constitutive expression of PoPUF60 expression occurred in relatively high levels in the heart and liver of flounder.E.tarda infection upregulated PoPUF60 expression,whereas RBIV-C1 infection downregulated PoPUF60 expression.Interference with PoPUF60 expression or overexpression of pol-miR-novel＿395 in flounder cells strongly potentiated E.tarda infection.Consistently,in vivo knockdown of PoPUF60 enhanced bacterial dissemination in the tissues of flounder but blocked viral replication,whereas in vivo overexpression of PoPUF60 inhibited bacterial dissemination but facilitated viral replication.In summary,we obtained the first expression profile of E.tarda-induced host miRNAs expressed in Japanese flounder kidney,and revealed the regulatory mechanisms of pol-miR-182-5p,pol-miR-novel＿171,pol-miR-novel＿547 and pol-miR-novel＿395 and their effects on pathogen infection,cellular autophagy,and cellular apoptosis.These results provided new insights into the mechanism of miRNA-mediated flounder immune defense against bacterial infection.