A Study On The Expression,Regulation,and Function Of Streptococcus Iniae-Induced MicroRNAs Of Paralichthys Olivaceus

Streptococcus iniae is a serious bacterial pathogen in marine fish.The outbreak of S.iniae disease has brought huge economic losses and severely hampered the development of fish farming industry.Now,more and more researchers have been studying the immune response mechanism of marine fish,which will provide theoretical and scientific basis for the prevention and treatment of bacterial disease in fish.MicroRNAs(miRNAs)are involved in many biological activities including immune defense against pathogens.In this study,we applied high-throughput sequencing technology to examine miRNAs in Japanese flounder(Paralichthys olivaceus)infected with S.iniae at different times.A total of 1038 miRNAs were identified,of which,249 were novel miRNAs,and 81 showed differential expression(named DEmiRNAs)after S.iniae infection.Of the 81 DEmiRNAs identified,34 and58 occurred at 6 h and 24 h post-infection,respectively;most DEmiRNAs were strongly time-specific,and only 13.6% of the DEmiRNAs were shared between the two time points.A total of 9582 target genes were predicted for the 81 DEmiRNAs.The putative target genes were enriched in various GO and KEGG pathways of biological processes and molecular/cellular functions,in particular endocytosis,regulation of transcription,lysososme,and the signaling pathways of MAPK,Erb B,and AMPK.Two of the DEmiRNAs,pol-3p-10740?175 and pol-miR-216 b were chosen for further study.pol-3p-10740?175 was found to target dual specificity phosphatase 6(Dusp6)and repress the expression of Dusp6.Transfection of flounder FG cells with pol-3p-10740?175 caused a significant inhibition on S.iniae invasion.q RT-PCR analysis showed that pol-miR-216 b expression was regulated by S.iniae and megalocytivirus in a pathogen-specific manner,and that pol-miR-216 b targeted the gene of FAIM1(Fas apoptotic inhibitory molecule,transcript variant X1,FAIM1)of flounder(named PoFAIM1)by negative interaction with the 3'-UTR of FAIM1.Fas apoptosis inhibitory molecule(FAIM)is a protein identified as an antagonist of Fas-induced cell death.However,its working mechanism and role in fish are unknown.We found that PoFAIM1 is highly conserved with mammalian FAIMs in structure and has a FAIM1 domain.To investigate the function of PoFAIM1,we constructedRNA interfering(RNAi)plasmids that expresses a small interferingRNA targeting PoFAIM1.Administration of flounder with theRNAi plasmids caused a significant inhibition on the expression level of PoFAIM1 in the liver,spleen,kidney and intestine,indicating that these plasmids can be used to knockdown the expression of PoFAIM1 in vivo.The results of this study provided the first S.iniae-induced miRNA profile in Japanese flounder,identified a large number of S.iniae-induced host miRNAs and demonstrated that flounder miRNAs play an important role in antibacterial immunity.These results added new insights into the function of fish miRNAs in the immune defense against bacterial infection.