The Function Of LncRNA In Megalobrama Amblycephala After Infection By Aeromonas Hydrophila

Long non-coding RNA(lnc RNA)is a kind of non-coding RNA(nc RNA)longer than 200 nt.lnc RNA plays a variety of roles in biological processes,including participating in the regulation of organelle function and metabolism,regulating the mediation regulation of cell signaling in cancer,and regulating the immune response to bacterial infection.In recent years,there have been many in-depth studies on mammalian lnc RNA,but the relevant studies on fish are very limited.Meanwhile,since lnc RNA is not conserved among species,it is difficult to apply the existing results directly to unstudied species.lnc RNA can interact with DNA,RNA,and proteins to regulate gene expression and is an important non-coding RNA that critical in the immune response process.Megalobrama amblycephala is one of the most economically important freshwater fish in China,and the bacterial septicemia caused by Aeromonas hydrophila is a serious threat to the breeding industry of M.amblycephala.Unfortunately,the characterization of M.amblycephala lnc RNA in response to A.hydrophila infection has not been performed.To better understand the biological significance of lnc RNA in the immunity system,the fish liver at different time points after A.hydrophila infection were obtained for lnc RNA-sequencing(lnc RNA-seq).Based on the different challenge time,the association analysis of m RNA and lnc RNA expression patterns using weighted gene co-expression network analysis(WGCNA)was conducted to uncover a large number of key m RNA and lnc RNA related to immunity.Subsequently,by overexpressing lnc RNA MSTRG.59348.1,MSTRG.5748.1 and MSTRG.7894.1,cell localization,quantitative PCR(q PCR),CCK-8,RNA pull-down and protein profiling were used to verify the function of lnc RNA,target genes and interacting proteins.The main results are as follows:1.Based on lnc RNA-seq analysis a total of 14,849 lnc RNA were identified,and2,196 lnc RNA showed significant differences at 0,4,12,24,and 72 hour post A.hydrophila infection(hpi).Gene Ontology(GO)annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analyses showed that the target genes of the differentially expressed lnc RNA were enriched in several pathways related to immune such as apoptosis,inflammation,and immune response.Furthermore,immune-related genes and lnc RNA in the time-specific modules were subsequently verified by q PCR.Time-specific modules were then identified,using WGCNA,and 28 modules significantly correlated with different time points after infection were found.2.After separated the nucleus and cytoplasm of the hepatocytes from M.amblycephala,cellular localization of three selected immune-related lnc RNA was performed to predict their functions.The results showed that MSTRG.59348.1 and MSTRG.5748.1 were mainly distributed in the nucleus,suggesting their functions are mostly to regulate the expression of downstream genes through epistasis and transcription.MSTRG.7894.1 was existed in both the nucleus and cytoplasm,which indicates that it has many regulatory modes.q PCR analysis showed that the three lnc RNA were expressed in the immune-related organs of M.amblycephala,and significantly changed in the liver after A.hydrophila infection.RNA-seq analysis of muscle cells overexpressing lnc RNA MSTRG.59348.1,MSTRG.5748.1 and MSTRG.7894.1 revealed that these genes were mainly enriched in antigen processing and presentation via MHC class I,RLRs signaling,IFN-related pathway enrichment,and a large number of pathway-related genes were significantly downregulated after lnc RNA overexpressed in muscle cells of M.amblycephala.Overexpression of lnc RNA significantly inhibited the expression of STING and IFN,and significantly upregulated muscle cell viability and promoted cell proliferation by targeting STING and IFN.3.The m RNA levels of MSTRG.59348.1 and MSTRG.5748.1 were significantly up-regulated after LPS stimulation,but MSTRG.59348.1 showed a stronger inflammatory response.RNA pull down experiments on MSTRG.59348.1 screened 57RNA-binding proteins(RBPs).The results of GO and KEGG analyses showed that MSTRG.59348.1 target proteins were enriched in immune-related pathways,such as peroxisome,drug metabolism,retinol metabolism and salmonella infection.lnc RNA can directly regulate the gene expression of epigenetically related RBPs,such as RPL22L1,RPL29,tubulin and NEDD8.RPL29 was identified as an RBP of lnc RNA MSTRG.59348.1.RPL29 was cationic and amphiphilic,and the N-terminal amino acid sequence is highly conserved among different species,laying the foundation for further studies of natural antimicrobial peptides in teleost fish.