Exploring The Mechanisms Of Liver Fibrosis Through Transcriptomics And Proteomics O F Hepatic Stellate Cells And Hepatocytes

Background and Objectives:Liver fibrosis is a pathological progression characterized by dysregulation of extracellular matrix production and degradation when liver cells undergo necrosis and inflammation.It ultimately leads to cirrhosis,particularly common in the Asia-Pacific region due to HBV infections.Timely and effective antiviral therapy following HBV infection can often halt fibrosis progression,preventing end-stage liver disease.Therefore,understanding the mechanisms underlying liver fibrosis and its reversal is of paramount importance.Studies have demonstrated the pivotal role of hepatic stellate cells(HSCs)in the development of liver fibrosis,where sustained activation of these cells is a key driving factor.Additionally,hepatocytes play a significant role during liver fibrosis by generating an inflammatory response when affected by factors such as viral infection,lipidosis,or alcohol toxicity.This response recruits pro-inflammatory monocytes and macrophages to the site of injury,releasing cytokines and chemokines that activate HSCs.Transcriptomics and proteomics have made significant strides in liver disease research.Various molecular markers associated with liver fibrosis have been identified,shedding light on its mechanisms.However,prior omics studies often relied on animal models or liver tissue samples from clinical patients,which might not fully represent the clinical progression of liver fibrosis.This study proposes the application of primary human liver cells,including hepatocytes and HSCs isolated from human liver tissue,combined with RNA sequencing and mass spectrometry(MS)techniques to investigate the expression profiles of these cells during the development of hepatitis B-related liver fibrosis.The aim is to identify significantly altered m RNAs and proteins associated with advanced liver fibrosis and cirrhosis,thus elucidating the pathogenesis and exploring potential molecular targets for anti-fibrotic therapies.The primary objectives of this research include:1.Utilizing transcriptomics and proteomics to analyze changes in m RNA and protein expression in hepatocytes and HSCs from liver tissue samples of cirrhotic patients and controls,identifying differentially expressed genes and proteins associated with liver fibrosis.2.Employing bioinformatics methods such as GO enrichment,KEGG pathway analysis,and protein-protein interaction(PPI)network analysis to elucidate the signaling pathways related to hepatocyte and HSC activation in liver fibrosis.Experimental validation of these findings will be performed.3.Integrating bioinformatics analyses of transcriptomic and proteomic data to provide a comprehensive understanding of the molecular mechanisms underlying hepatitis B-related liver fibrosis.Experimental Methods:1.Collection of discarded liver tissue samples from patients undergoing liver transplantation or partial hepatectomy at Jilin University First Hospital.Liver tissue blocks with intact blood vessels and liver capsules will be selected and processed.Primary hepatocytes and HSCs will be isolated from these tissues and validated.2.Total RNA will be extracted from isolated cells,and c DNA libraries will be constructed.Sequencing of samples will generate raw FASTQ data,followed by differential gene expression analysis,resulting in the identification of differentially expressed genes.GO enrichment,KEGG pathway analysis,and PPI network analysis will be performed.3.Isolated cells will undergo high-p H reverse-phase fractionation(HPRP)and liquid chromatography-tandem mass spectrometry(LC-MS/MS)analysis in DDA mode to build a library.Subsequently,samples will undergo LC-MS/MS analysis in DIA mode using the DDA library for qualitative and quantitative analysis.Bioinformatics analysis will be conducted on the post-sequencing data,including identification,expression analysis,GO enrichment,KEGG pathway analysis,and PPI network analysis.4.Combining transcriptomic and proteomic data,overlapping differentially expressed proteins will be identified,and their interactions will be analyzed.Candidate molecules,such as PKM2 and EHD2,which are up-regulated in the transcriptome and proteome of HSCs,will be validated through RT-q PCR,Western blot,and immunofluorescence.Functional studies on these molecules will be conducted in the LX-2 cell line.Experimental Results:1.Transcriptomic Analysis: In HSCs,a total of 2,156 differentially expressed genes were identified,comprising 1,040 up-regulated and1,116 down-regulated genes.Bioinformatics analysis revealed that differentially transcribed genes were associated with small molecule catabolism,wound healing pathways,extracellular matrix composition,carboxylic acid catabolism,and tissue acid catabolism.They were predominantly enriched in collagen extracellular matrix and mitochondrial matrix.KEGG analysis showed enrichment in focal adhesion,retinol metabolism,drug metabolism by cytochrome P450,tyrosine metabolism,and pentose and glucuronate interconversions.In HCs,2,994 differentially expressed RNAs were identified,consisting of1,395 up-regulated m RNAs and 1,599 down-regulated m RNAs.Bioinformatics analysis revealed enrichment in positive regulation of cytokine production,positive regulation of leukocyte adhesion,myeloid leukocyte activation,peptide antigen presentation,collagen extracellular matrix,mitochondrial matrix,actin filament,basement membrane,collagen trimer,MHC complex,actin binding,extracellular matrix structural constituent,transmembrane transporter binding,peptide antigen binding,and ethanol dehydrogenase activity.KEGG analysis showed enrichment in cell adhesion molecules and osteoclast differentiation pathways.2.Proteomic Analysis: In HSCs,711 differentially expressed proteins were identified,including 643 up-regulated and 68down-regulated proteins.Bioinformatics analysis indicated that differentially expressed proteins were primarily involved in cell adhesion,myofibril assembly pathways,positive regulation of leukocyte activation,positive regulation of cell activation,and protein complex assembly regulation.They were mainly enriched in cell basement membrane,cell adhesion,secretory granule membrane,phagocytic vesicle,and actin filament bundle.KEGG analysis revealed enrichment in phagosome,tuberculosis,NK cell-mediated cytotoxicity,leukocyte transendothelial migration,and FcγR-mediated phagocytosis.In HCs,1,711 proteins were identified,comprising 1,311 up-regulated proteins and 400down-regulated proteins.Bioinformatics analysis showed that differentially expressed proteins were predominantly enriched in phagocytosis,antigen presentation processes,vacuolar membrane,lysosomal membrane,soluble vacuolar membrane,collagen extracellular matrix,endocytic vesicle,integrin binding,MHC binding,peptide antigen binding,and carbohydrate kinase activity.KEGG analysis revealed enrichment in phagosomes,cell adhesion molecules,glycolysis,and fructose and mannose metabolism.3.Integration of Transcriptomics and Proteomics: Matching analysis of differentially expressed proteins and genes in hepatic stellate cells(HSCs)revealed 96 up-regulated and 14 down-regulated proteins.Joint analysis of transcriptomics and proteomics in liver cells yielded 53 overlapping differentially expressed proteins,including 34 up-regulated and 19 down-regulated proteins.Protein-protein interaction(PPI)analysis of the 106 overlapping proteins in HSCs identified a network of 76 interacting proteins.Subsequent analysis of associated factors and literature review highlighted increased expression levels of PKM2 involved in glycolysis and EHD2 regulating membrane folding function in both primary human HSCs and TGF-β-stimulated LX-2 cells.This was further validated through RT-q PCR,Western blot,and immunofluorescence.Overexpression of PKM2 and EHD2 promoted collagen production in hepatic stellate cells.Conclusion:1.Integrated transcriptomic and proteomic analysis revealed 110 overlapping proteins in HSCs,of which 96 were up-regulated and 14 were down-regulated,suggesting their involvement in focal adhesion,cell-matrix interaction,leukocyte transendothelial migration,NK cell-mediated cytotoxicity,amino sugar and nucleotide sugar metabolism,and other pathways.2.Significant differences in transcriptomics and proteomics between liver cirrhosis and control groups were identified.Integrated analysis highlighted 53 overlapping differentially expressed proteins,functionally enriched in phagocytosis,antigen processing,integrin binding,MHC binding,peptide antigen binding,autophagosome,cell adhesion molecules,glycolysis,and fructose and mannose metabolism,among others.3.During liver fibrosis,PKM2 and EHD2 exhibited significantly increased m RNA and protein expression levels in HSCs.Overexpression of PKM2 and EHD2 promoted collagen production in HSCs.These findings suggest that PKM2 and EHD2 may play pivotal roles in the pathogenesis of liver fibrosis,potentially serving as therapeutic targets.The study provides new insights into HBV related liver fibrosis through integrated analysis of transcriptomics and proteomics methods and reveals that PKM2 and EHD2 may play important roles in liver fibrosis,which can help further elucidate the mechanism of liver fibrosis or become targets for liver fibrosis treatment.