Transcriptomics Research On Cortisol-induced Hepatocytes Injury In Grouper(Epinephelus Coioides)

In this experiment,the effects of cortisol on hepatocytes and its action mechanism in Epinephelus coioides were investigated by using the grouper hepatocyte primary culture method and transcriptomics technique.The cortisol on grouper liver cells damage mechanism was revealed from the level of transcriptomics.The main experimental results showed as follows:1、Effect of cortisol on primary hepatocytes function of grouperBased on the experimental results obtained before the laboratory,two groups of cortisol level(0,1000nmol/L)were used to incubate hepatocytes for 36 hour to study the effect of cortisol on the primary hepatocytes function of the grouper(Epinephelus coioides).The two groups of hepatocyte culture supernatants were tested for aspartate aminotransferase.(AST),alanine aminotransferase(ALT),lactate dehydrogenase(LDH),alkaline phosphatase(AKP)activity,and intracellular malondialdehyde(MDA)content,and found that the cortisol group compared with the normal group,AST ALT,LDH,AKP activity and MDA content were significantly increased(P<0.05).Total cholesterol(T-CHO),total bilirubin(TBIL),triglyceride(TG)content and total antioxidant capacity(T-AOC)were measured in the supernatants of hepatocytes of the two groups,and showed that the levels of T-CHO,TBIL and TG were significantly higher in the cortisol group than in the normal group(P<0.05).What’s more,in the cortisol group of T-AOC was also compared with the control group was significantly improved(P<0.05).The results demonstrated that 1000 nM cortisol had damage effect on the liver cells of the grouper(Epinephelus coioides).2、Transcriptome analysis of hepatocytes injury induced by cortisol in grouperThe transcriptome sequencing of two groups of grouper hepatocytes samples was carried out on the basis of experiment 1.The transcriptome results showed that six cDNA libraries were constructed from two groups of hepatocyte samples.56.64 millions,54.40 millions,98.29 millions,81.85 millions,78.14 millions and 87.09 millions clean reads were obtained in the six cDNA libraries(cortisol2,cortisol3,cortisol5,normal2,normal5,normal6),respectively.A total of 112,618 unigenes were gained by splicing and assembling in the six cDNA libraries,with an average length of 957.45 bp,a maximum length of 26793 bp,a minimum length of 201 bp,GC percentage of 44.19%,N50 values of 1687 bp and N90 values of 311 bp.The unigenes were compared with Nr,Pfam,Swiss-prot,String,GO,KEGG and other databases for BLASTx similarity founded that a total of 110560(98.17%)unigenes annotated 1748 significant differentially expressed unigenes(1277 up-regulated genes,471 down-regulated genes),which were screened by gene expression comparison analysis.Differential expression genes(DEGs)GO and KEGG enrichment analysis indicated that cortisol-induced hepatocytes injury mainly caused intracellular signal transduction,post-translational modification,protein turnover,catalytic activity,stimulation response and other biological regulation processes.DEGs were mainly enriched in a series of physiological metabolic regulation pathways,such as complement and coagulation cascade pathway,cytokine-cytokine receptor pathway,alanine,aspartic acid and glutamate metabolic pathway,AMPK signaling pathway,insulin signaling pathway,glycolysis/gluconeogenesis pathway,hematopoietic lineage,carbon metabolism and bile acid secretion.It manifested that these DEGs were likely to involved in the cortisol-induced injury of hepatocytes,participated in the immune response of various signaling pathways of cells,and played multiple joint effect role in the hepatocytes injury process regulated by various transcription factors.The 45 differentially expressed genes,involved in Toll-like receptor signaling pathway,complement and coagulation cascade,hematopoietic lineage,bile secretion pathway,and phosphatidylinositol 3-kinase/protein kinase signaling pathway,were used to verify the reliability of the transcriptiome data by RT-qPCR.Based on the above results,it found that GR signaling pathway,PI3k-Akt signaling pathway,Toll-like receptor signaling pathway and bile secretion were involved in the process of cortisol-induced injury of grouper liver cells;PI3k-Akt signaling pathway and Toll-like receptor played an important role among them.3、Expression profile of LncRNA in cortisol-induced injury of hepatocytes in grouperThis study analyzed the changes in the expression profile of LncRNAs in the process of grouper’s hepatocytes injury induced by cortisol.The lncRNA sequencing analysis showed that 9687 new lncRNAs and 314 known lncRNAs were predicted by bioinformatics methods.A total of 540 lncRNAs with significant differences were screened by padj<0.05&|log2FC|>1 screening conditions,which 150 lncRNAs significantly down-regulation and 390 significantly up-regulation lncRNAs.The reliability of lncRNA sequencing results were confirmed by selecting 33 differential expression lncRNAs for RT-qPCR verification(r=0.6933,p<0.01).Differential expression lncRNA target genes were mainly enriched in cytokine-cytokine receptor signaling pathway,PI3k-Akt signaling pathway,complement and coagulation cascade,hematopoietic lineage,HIF-1 signaling pathway,Toll-like receptor signaling pathway and bile acid secretion in the injury process of hepatocytes induced by cortisol.These results further illustrate that lncRNAs play an important role mainly by participating in PI3k-Akt signaling pathway and Toll-like signaling pathway.