Study On The Hepatotoxicity Mechanism Of MC-LR In Silver Carp By Transcriptome Analysis

In the recent years,the cyanotoxin MC-LR has received much more concerns by the researchers in Toxicology due to the fact that it can be toxic to animals and humans.However,the mechanism of MC-LR-hepatotoxicity in detail has not fully understood especially for silver carp.In the present study,the second-generation transcriptome sequencing was used to analyze the toxic effect of MC-LR on the liver of silver carp as to systemically understand the hepatotoxicity mechanism of MC-LR.Silver carp was used the experimental animal in this study and the 24 h LD₅₀ of MC-LR in the fish was obtained by the method of Karber.According to the obtained LD₅₀ in the acute toxicity test,the fish were divided into three groups in which two groups（50 and 200 μg/kg of MC-LR）were used as treatment groups and one group as the control.The treated fish received 50 and 200 μg/kg of MC-LR,respectively by intraperitoneal injection and the control fish received the same volume of PBS.The fish was exposed to MC-LR for 24 h and then they were sacrificed and their livers were taken and used for total RNA extraction.The total RNA was isolated from the livers using an RNAiso Plus kit according to manufacturer’s instructions.Total RNA concentration and purity were determined spectrophotometrically according to the method of Sambrook and Russel.Totally 30 μg of RNA was mixed with 3 independent-treatment groups as one sample and then send to BGI（Huada Genomics Institute Co.Ltd,China）for deep sequencing using Illumina HiSeq 4 000 platform.Raw sequence reads were filtered by the Illumina pipeline.The 3’ adaptor sequence was removed from raw sequences and all low-quality tags such as short tags,empty reads,and singletons（tags that occurred only once）were removed.The remaining high-quality sequences were mapped to human genome（ftp://ftp.ncbi.nlm.nih.gov/genomes/Homo_sapiens/Assembled_chromosomes/）using SOAP algorithm.The digital expression level of each annotated gene was calculated using the RPKM method（Reads per kilobase transcriptome per million mapped reads）.The GO functional analysis of the differential expressed genes（DEGs）was performed by GO program（www.geneontology.org）to identify the principle biological functions and the KEGG pathway（http://www.genome.jp/kegg/）analysis was performed in order to identify DEGs with similar functions.Finally,the expressions of major DEGs were determined and verified by qPCR.The main results obtained in this study were as following.（1）The result of De novo sequencingAfter deep sequencing,the mean length of the transcripts of 53 611 308,54 217 023,and 66 980 128 bp raw reads were obtained from the control,50,and 200 μg/kg MC-LR treatment groups,respectively,in which the mean length of the transcripts were 790,779,and 830 bp,respectively.After the assembly,the number of Unigenes were 50 116,58 987,and 49 383 in three groups,respectively.According to the result of statistics,the DEGs in 50 μg/kg MC-LR group were 2 565 in up-regulation and 1 831 in down-regulation when compared to the control.While in 200 μg/kg MC-LR group,they were 7 256 in up-regulation and 4 751 in down-regulation.（2）The results of GO analysisThe differential expression genes were performed in the GO database for comparison analysis one by one and made a comment on the function of the gene and the participation of biological processes.The results indicated that the GO population was distributed between zebrafish and grass carp.A total of 14 268 DEGs were identified in the GO database with a coverage rate of 20.67%.The DEGs in the MC-LR-treated liver of silver carp were mainly enriched in the organelles,nucleus,cell membrane,macromolecule complex,cell connection,etc.These DEGs might affect the activity of the receptor,the binding activity of the protein to the transcription factor,transcriptional activity,molecular transduction activity,enzyme moderator activity,catalytic activity and other oxidation and reduction processes,organic acid metabolism,organic nitrogen compounds metabolism,and coenzyme factor metabolism,in which DNA damage repair process was significantly enriched The main participation involved might be reaction,production and reproduction,biological positive/negative regulation,redox,metabolic process,hormone secretion,immune system,cell tissue or biogenesis,cell aggregation,and cell adhesion processes.（3）The results of KEGG analysisThe results of KEGG pathway analysis showed that a total of 31 890 Unigene were identified in the KEGG database with a coverage rate of 46.19%.The mechanisms of MC-LR-hepatotoxicity may be mainly involved in NF-κB signaling pathway,p53 signaling pathway,phosphatidylinositol 3 signaling pathway,Wnt signaling pathway.（4）The result of qPCRThe results of qPCR verification showed that the expressions of the main DEGs Raf1,CDC42/RAS,ERK,c-jun,c-myc,p53,S6K1/2,SOS,PTEN,AKT,IKKα,IKKβ,caspase-8,bax,P21,SHP,RAC,and PP2 A were up-regulated in the MC-LR-treated groups when compared to the control group,which is consistent with the predicted results by sequencing.In conclusion,the molecular mechnism of MC-LR-hepatotoxicity in silver carp was invested aged by De novo sequencing and transcriptome analysis in the present study.Moreover,important DEGs and the involved pathways in MC-LR-hepatotoxicity were identified.This study may be useful for revealing the toxicity mechanism theory of MC-LR,finding new drug target for liver disease prevention,and evaluating the possible risk of MC-LR on human health.