A gene-based clustering approach reveals QSOX1/IL1RAP as promising biomarkers for the severity of non-alcoholic fatty liver disease

Abstract

Background and Aims: Non-alcoholic fatty liver disease (NAFLD) is a progressive liver disease that ranges from simple steatosis to inflammation, fibrosis, and cirrhosis. To address the unmet need for new NAFLD biomarkers, we aimed to identify candidate biomarkers using publicly available RNA sequencing (RNA-seq) and proteomics data. Methods: A novel approach involving unsupervised gene clustering was performed using homogeneously processed and integrated RNA-seq data of 625 liver specimens to screen for NAFLD biomarkers, in combination with public proteomics data from healthy controls and NAFLD patients. Additionally, we validated the results in the NAFLD and healthy cohorts using enzyme-linked immunosorbent assay (ELISA) of plasma and immunohistochemical staining (IHC) of liver samples. Results: We generated a database (https://dreamapp.biomed.au.dk/NAFLD/) for exploring gene expression changes along NAFLD progression to facilitate the identification of genes and pathways involved in the disease's progression. Through cross-analysis of the gene and protein clusters, we identified 38 genes as potential biomarkers for NAFLD severity. Up-regulation of Quiescin sulfhydryl oxidase 1 (QSOX1) and down-regulation of Interleukin-1 receptor accessory protein (IL1RAP) were associated with increasing NAFLD severity in RNA-seq and proteomics data. Particularly, the QSOX1/IL1RAP ratio in plasma demonstrated effectiveness in diagnosing NAFLD, with an area under the receiver operating characteristic (AUROC) of up to 0.95 as quantified by proteomics profiling, and an AUROC of 0.82 with ELISA. Conclusions: We discovered a significant association between the levels of QSOX1/IL1RAP and NAFLD severity. Furthermore, the QSOX1/IL1RAP ratio shows promise as a non-invasive biomarker for diagnosing NAFLD and assessing its severity. (ChiCTR2300073940).

Unveiling the pathogenesis of non-alcoholic fatty liver disease by decoding biomarkers through integrated single-cell and single-nucleus profiles

“…To address this question, we performed immunohistochemical staining (IHC) for liver QSOX1/IL1RAP on a group of NAFLD patients from Shenzhen Traditional Chinese Medicine Hospital, China (SZTCMH). Details of this cohort have been provided previously (16).…”

“…For IHC, we collected 12 fixed liver tissues from archived histological samples at SZTCMH between 2014 and 2023, and details were in our earlier study (16).…”

“…In our previous study (16), we pinpointed key genes that are significantly involved in the advancement of NAFLD. Specifically, we discovered 16 genes, including A2M, C7, COL6A3, COLEC11, ENPP2, FBLN1, FBN1, FCGBP, IGFBP6, LCN2, LUM, MMP2, PAPLN, PTGDS, QSOX1, and VWF, that exhibited elevated secretory activity correlating with the severity of NAFLD.…”

“…Recently, we demonstrated that Quiescin sulfhydryl oxidase 1 (QSOX1) and Interleukin-1 receptor accessory protein (IL1RAP) hold potential as indicators of liver disease severity in NAFLD patients (16). QSOX1, a flavin-linked enzyme that facilitates disulfide bond formation within the secretory structures of mammalian cells, is expelled into the extracellular matrix (ECM) and blood as an independent protein through the standard secretory pathway, encompassing the Golgi apparatus, secretory vesicles, and the plasma membrane.…”

“…QSOX1, a flavin-linked enzyme that facilitates disulfide bond formation within the secretory structures of mammalian cells, is expelled into the extracellular matrix (ECM) and blood as an independent protein through the standard secretory pathway, encompassing the Golgi apparatus, secretory vesicles, and the plasma membrane. Furthermore, it is notably amplified and released from fibroblasts that are actively synthesizing ECM precursors (15)(16)(17).…”