Identification And Characterization Of Liver CircRNA And Immune Regulatory Network Analysis Of Megalobrama Amblycephala After Aeromonas Hydrophila Infection

Blunt snout bream(Megalobrama amblycephala)is the sixth largest freshwater cultured fish in China,and bacterial septicemia caused by Aeromonas hydrophila has caused severe damage to the M.amblycephala aquaculture industry.Circular RNAs(circRNAs),a class of non-coding RNA,play an important role in regulating various biological processes.It has been reported that circRNAs play an important regulatory function in non-specific immune responses in mammalian species,while there are few studies in fish,and only a few circRNAs has been identified in fish.In order to explore the immune regulatory function of circRNA in the process of A.hydrophila infection,the circRNA expression pattern in the M.amblycephala liver was identified at different time points post A.hydrophila infection using RNA-seq technology in this study,and the function of circRNA was comprehensively analyzed and predicted by difference analysis,gene function enrichment analysis,ce RNA network construction,weighted gene co-expression network analysis(WGCNA)and other methods,and the results were further verified by q PCR,overexpression and inhibition,dual-luciferase reporter assays and other experimental methods.The results of this study will enrich the circRNA information of teleost,and help to understand the non-specific immune regulation mechanism of the host from the perspective of non-coding RNA,and lay the foundation for effective control of pathogenic infection and genetic improvement of fish disease resistance in the future.The main results of this study are as follows:1.A total of 15 liver circRNA librairies were constructed at 5 time points before(0 h)and after(4 h、12 h、24 h and 72 h)A.hydrophila infection,and a total of163.21 Gb clean data were generated,with the genome alignment rate of 15 sequencing libraries ranging from 87.12% to 94.84%.A total of 250 circRNAs were identified by find＿circ and CIRI,the average length of circRNAs was 305 bp,61.2%circRNAs were expressed at all five time points,and more than 50% circRNAs were derived from exons.These circRNAs were distributed on all the 24 chromosomes without obvious distribution pattern.A total of 106 differentially expressed(DE)circRNAs were identified by comparing the circRNA expression profiles at 5 time points.The parental genes of these DE circRNAs were enriched in the complement and coagulation cascades,Fcγ-R mediated phagocytosis and chemokine signaling pathways,suggesting that these DE circRNAs may play an important role in immune regulation.In addition,PCR was used to verify the loop formation of 12 randomly selected circRNAs,and q PCR was used to verify the expression trend of 6 randomly selected circRNAs,and the results showed that the RNA-seq data were reliable.2.The prediction software found that 208 circRNAs had mi RNAs binding sites,which could bind to 162 mi RNAs,generating a total of 1172 circRNA-mi RNA targeting relationships.Combined with immune-related mi RNA-m RNA network,an immune-related ce RNA network in the liver of M.amblycephala was constructed,which included 139 circRNAs,39 mi RNAs and 72 immune-related m RNAs.The target genes in the network including interleukin,complement,interferon and chemokine,which play an important role in resistance to bacterial infection,indicating that circRNA is involved in immune regulation against bacterial infection.3.A total of 249 circRNAs and 7135 m RNAs were combined for WGCNA.According to the correlation of gene expression levels,a cluster tree was constructed,and modules were divided,and 13 co-expression modules were eventually identified,5 of these modules(lightpink4、darkgreen、salmon4、honeydew1 and plum1)were found to be highly correlated with A.hydrophila infection.Functional enrichment analysis showed that the functions of these specific modules were mainly related to pathogen recognition,chemokine activation,inflammatory response activation,oxygen transport,angiogenesis,iron ion homeostasis,coagulation and complement activation.These results were helpful to understand the function of circRNA in the module and the immune response mechanism of fish against bacterial infection.4.It was predicted that circPGAP2 and circ EPB41L2 contained mi R-146 a binding site,and the dual-luciferase reporter assay system was used to verify that circPGAP2 could target mi R-146 a.Divergent primer and convergent primer were used to verify that circPGAP2 was a circRNA.The expression level of circPGAP2 in blood was the highest,and subcellular location showed that circPGAP2 mainly existed in the cytoplasm.In conclusion,circPGAP2 may be involved in downstream immune gene regulation as a molecular sponge of mi R-146 a.5.It was predicted that circ ARHGEF15 and circ AKT2 contained mi R-214 binding site,and the dual-luciferase reporter assay system was used to verify that circ ARHGEF15 and circ AKT2 could target mi R-214.Subcellular location showed that circ ARHGEF15 and circ AKT2 were mainly located in the cytoplasm,indicating that circ ARHGEF15 and circ AKT2 had the foundation to become mi RNA sponges.Furthermore,the dual-luciferase reporter assay system demonstrated that mi R-214 could target hepcidin.After overexpression and inhibition of mi R-214 in L8824 cells,the expression of hepcidin decreased and increased accordingly.