Transcriptomic and Proteomic Analyses Identify THBS2, VWF, NFASC, and AKR1B10 as Diagnostic Biomarkers for Advanced Fibrosis in NAFLD

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Purpose: The increasing prevalence of non-alcoholic fatty liver disease (NAFLD) has paralleled changes in lifestyle and dietary habits. However, the gold standard for the staging of NAFLD relies on liver biopsy, which is highly damaging and poorly accepted. This study aims to identify convenient biomarkers for staging advanced fibrosis (stage F3–F4) in NAFLD patients, thereby improving disease management. Patients and Methods: This study integrated liver transcriptomic sequencing data and peripheral blood proteomic data from NAFLD patients. Candidate diagnostic biomarkers for advanced fibrosis were screened using three machine learning algorithms. The dynamic expression patterns of these biomarkers were further validated by time series analysis from NAFLD mouse model. Additionally, a retrospective validation cohort comprising 119 biopsy-confirmed NAFLD patients was established at Shandong Provincial Hospital. Blood samples and clinical characteristics were collected before liver biopsy. These samples were used to validate the diagnostic performance of the candidate biomarkers via ELISA, which was further compared with established non‑invasive indicators (FIB‑4, APRI, and NFS). Finally, single-nucleus sequencing was employed to elucidate the underlying pathogenic mechanisms and their cellular location in NAFLD, while Mendelian randomization (MR) analyses based on whole proteome and whole genome data were conducted to explore causal relationship for NAFLD. Results: This study identified THBS2, AKR1B10, NFASC, and VWF as diagnostic biomarkers for advanced fibrosis in NAFLD. Transcriptomic and proteomic analyses demonstrated that the combination of these four biomarkers exhibited robust diagnostic performance (AUC = 0.860– 0.880). In our NAFLD clinical cohort, each biomarker individually showed promising diagnostic performance (THBS2 AUC = 0.867; NFASC AUC = 0.834; VWF AUC = 0.777; AKR1B10 AUC = 0.733), which was comparable to established non-invasive indicators. Furthermore, the expression levels of THBS2, AKR1B10, and VWF increased progressively with the duration of high-fat diet feeding in NAFLD mouse model. NFASC and THBS2 were specifically enriched in activated hepatic stellate cells and showed significant upregulation in advanced fibrosis and cirrhosis. Conclusion: THBS2, AKR1B10, NFASC, and VWF serve as diagnostic biomarkers for advanced fibrosis in NAFLD, providing novel insights for the development of non-invasive clinical diagnostic strategies. Diagram of NFALD patient analysis using biopsy, snRNA-seq, transcriptomics and proteomics for fibrosis diagnosis.The diagram outlines the analysis process for non-alcoholic fatty liver disease (NFALD) patients with varying fibrosis levels. It starts with a biopsy analyzed via single-nucleus RNA sequencing (snRNA-seq), enhancing macrophage and hepatic stellate cell interactions and immune infiltration. Transcriptomics and proteomics are performed, using weighted gene co-expression network analysis (WGCNA) and identifying differentially expressed genes (DEGs) and proteins (DEPs). This analysis identifies diagnostic biomarkers for advanced fibrosis, such as THBS2, AKR1B10, NFASC and VWF. These biomarkers undergo further analysis using LASSO, random forest (RF) and support vector machine (SVM) methods. Proteome-wide and genome-wide Mendelian randomization (MR) provide insights into NFALD and non-alcoholic steatohepatitis (NAFLD/NASH). Keywords: advanced NAFLD fibrosis, diagnostic biomarkers, multi-omics sequencing, THBS2

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