Integrative proteomics and transcriptomics identify novel fibrosis-related biomarkers of patients with post-HBV hepatitis cirrhosis

Background: Hepatitis B virus (HBV) infection is an important public health burden. Chronic HBV infection always leads to chronic hepatitis, liver fibrosis and increases the incidence of liver cancer. However, the mechanism of liver fibrosis caused by persistent HBV infection remains unclear. Methods: In present study, we isolated and enriched primary human hepatic stellate cells (HSCs) from chronically HBV infected patients with liver cirrhosis. The differences expression of mRNA and protein were analyzed by combining proteomics, transcriptomics, and biological network analysis. Results: After bioinformatics analysis with transcriptomics and proteomics data, a total of 2,156 genes and 711 proteins were differently expressed between the liver cirrhosis group and control group. GO enrichment analysis and KEGG analysis indicated those differently expressed molecules were mainly involved in oxidative stress, glycometabolism, and fibrous repair. Moreover, a total of 110 overlapping biomarker were identified and two of them (PKM2 and EHD2) were validated as candidates correlated with liver fibrosis by qRT-PCR and immunofluorescence from primary human HSCs and in vitro cellular hepatic fibrosis model. Conclusion: Our results indicated that PKM2 and EHD2 were overexpressed in chronically HBV infected patients with liver cirrhosis, suggesting them as potential biomarkers and therapeutic targets for liver fibrosis. Overall design: Liver biopsy samples were obtained from patients who admitted to the first hospital of Jilin university between 2020 to 2021. Chronically HBV infected patients with liver cirrhosis underwent liver transplantation were defined as cirrhosis group and hepatic hemangioma partial hepatectomy were defined as control group. All HBV-infected patients were diagnosed with the criteria recommended by the Asian Pacific Association for the Study of the Liver (APASL). Primary human HSCs were isolated from resectable liver sample using an established method.Total RNA was extracted from primary human HSCs with the RNeasy Mini kit (Qiagen, Germantown, Maryland, USA) according to manufacturer's protocol.RNAsamples were on sequenced Illumina Novaseq 6000 (Illumina, , California, USA).Protein preparation for each sample was performed as previous described.Proteomic analysis was performed by DDA mass spectrometry assay. The microarray datasets were processed with rigorous quality control procedures to ensure the highest possible level of data quality using the R 4.2.1 and the R package of “Bioconductor”.Through the “ClusterProfiler” package in R, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to discover the biological function and significant signaling pathways correlated with liver fibrosis.To identify more key genes related to liver fibrosis, a PPI network of differently expressed molecules was established by the online tool STRING (STRING, http://string-db.org) for exploring interactions between genes or proteins.