Proteomics And Transcriptomics Studies Of HBV-associated Liver Fibrosis

BackgroundLiver fibrosis is a worldwide disease with high morbidity and high mortality.In recent years,the study of liver fibrosis has become a hot field in liver disease research,due to the reversibility of liver fibrosis.Hepatitis B virus(HBV)infection is an important factor in causing chronic liver disease and is closely related to the occurrence and development of hepatic fibrosis.Although significant progress has been made in the study of hepatic fibrosis,the mechanism of HBV persistent infection related to liver fibrosis remains unclear.Therefore,it is of great significance for the diagnosis and treatment of liver fibrosis to elucidate the mechanism.At present,proteomics and transcriptomics are widely used in the study of liver diseases.A variety of molecular markers have been identified,as well as the mechanism of liver fibrosis has been improved by using this omics approach.Additionally,RNA-seq,which is a high-throughput transcriptomic technique,has been widely used in disease related molecular research.However,there are few reports about the pathogenesis of HBV-related liver fibrosis by using this transcriptomic technique.Therefore,it will be helpful to further clarify the pathogenesis of liver fibrosis and screen differentially expressed proteins,which is of great significance for the diagnosis and treatment of hepatic fibrosis.Objects:1.Proteomic approach was used to analyze the changes of protein expression in HBV-related liver fibrosis mouse models.The differentially expressed proteins in liver tissues were identified.2.Transcriptomic approach was used to analyze the transcriptional changes of genes in HBV-related liver fibrosis mouse models.The differentially expressed genes in liver tissues were identified.3.A comprehensive analysis of proteomic and transcriptome datas was performed to illustrate the pathogenesis and pathophysiology of HBV related liver fibrosis.Methods:1.AAV8-1.2HBV was injected into C57BL/6 mice through tail vein.Mouse serum and liver tissue samples were collected and the change of HBV DNA and other liver fibrosis related indexes were detected at 1,3,and 6 months post injection.The pathological changes of liver tissue were analyzed by immunohistochemical staining.2.Mouse liver tissue samples were collected at 1,3,and 6 months post injection.Total proteins were extracted and analyzed by two-dimensional gel electrophoresis.The differentially expressed protein spots were identified by MALDI-TOF/TOF MS and compared with the UnProt database.Then GO,KEGG pathway and protein-protein interaction(PPI)analysis were carried out to further assess the roles of differentially expressed proteins in the process of liver fibrosis.3.Mouse liver tissue samples were collected at 1,3,and 6 months post injection.Then total RNAs were extracted and the cDNA libraries were established according to the standards of Illumina Hiseq sequencing.Then the Hiseq sequencing was performed by using the 2x150bp pairing ends(paired-end,PE).Sequencing results were analyzed for gene expression level,differential gene screening,Gene Ontology(GO),KEGG pathway and protein-protein interaction(PPI).4.A comprehensive analysis of the proteomic and transcriptome findings was performed to find overlapping differentially expressed proteins.Western Blot and RT-qPCR anaylsis were performed to verify the accuracy of the omics results.Based on bioinformatics analysis,the mechanism of HBV related fibrosis was further expounded.RT-qPCR and siRNA were used to investigate the inflammatory associated gene NIrp1 in LX-2(HSC cell line).Results:1.A higher concentration of HBV DNA can be detected in the HBV(+)mouse liver tissue and serum samples in 1,3,6 months post AAV8-1.2HBV injection.The mice showed viremia.Pathological sections and fibrosis indexes showed that there was no obvious acute inflammatory reaction in liver tissues.However,there was chronic inflammatory response and fatty degeneration in HBV(+)mouse liver tissues.Additionally,HBsAg and HBeAg were accumulated in HBV(+)mouse liver tissues.The liver has developed fibrosis.2.173 differentially expressed proteins were identified by proteomic analysis.The significant differentially expressed proteins were mainly clustered in reactive oxygen species and mitochondrial part.KEGG analysis indicated that the identified proteins were enriched in 19 pathways including NOD-like receptor signaling pathway,RIG-I-like receptor signaling pathway,apoptosis.PPI results indicated that the interacting proteins have important functions in the clusters of amino acid metabolism,immune regulation,and oxidative stress response.3.Based on the transcriptome analysis,213 and 109 genes were up-regulated and down-regulated in all the three HBV(+)mouse libraries,respectively.For the up-regulated genes,the highly enriched GO terms are associated with a response to the organic acid metabolic process and Drug metabolism-cytochrome P450 signal pathway.The highly enriched GO terms in the down-regulated genes are associated with lipid metabolic process and peroxisome.PPI results showed that PI3K/AKT,JaK/STAT signal pathway were abnormalities.4.The differentially modulated proteins were compared with the differentially transcribed genes and 28 overlapped proteins were found.The overlapped proteins were highly enriched in response to glutathione metabolism,redox reaction,innate immunity and Wnt signaling pathway.The transcription and expression of proteins(CAT,GSTP1,PRDX1,NXN,BLVRB and IDH1)involved in the regulation of oxidative stress were significantly changed.These proteins may be regulated by ubiquitination,and UBC may be an important regulatory protein.The expression of TGF-beta 1 could be inhibited by silencing Nlrp1 in the LX2 cells(HSC cell line).Conclusions:1.In this study,a mouse model with persistent replication of hepatitis B virus and liver fibrosis was established.The model demonstrates distinct features of chronic liver disease,including viremia,chronic inflammation,steatosis and fibrosis.These characteristics are more similar to liver fibrosis induced by hepatitis B virus infection in humans.Thus,it provides a good animal model for the study of the mechanism of chronic hepatitis B related liver fibrosis.2.In this study,HBV related fibrosis was analyzed at RNA and protein levels.The important role of oxidative stress in the process of hepatic fibrosis was elucidated.The regulatory effects of CAT,BLVRB,NXN,PRDX1,and IDH1 on oxidative stress were also revealed.This result lays a foundation for understanding the pathogenesis and development of HBV related fibrosis.3.In the preliminary study of the fibrotic mechanism,the expression of TGF-beta1 could be inhibited by silencing of NIrp1.