Identification of Ferroptosis and Autophagy-Related Diagnostic Biomarkers and Immune Infiltration in Non-Alcoholic Fatty Liver Disease Using Integrated Bioinformatics Analysis

Abstract

Aims: Non-alcoholic fatty liver disease (NAFLD) is a clinicopathological syndrome characterized by excessive deposition of fat in hepatocytes. This study aims to investigate the mechanisms of ferroptosis and autophagy-related diagnostic biomarkers and immune cell infiltration in NAFLD. 

Main methods: GSE89632 was explored to investigate the differentially expressed genes (DEGs). GO/KEGG pathway enrichment analysis was carried out using the Metascape and “clusterProfiler” Package of R. The STRING was used to obtain a PPI network. Hub genes were calculated to reflect the diagnostic accuracy of the gene by receiver operator characteristic (ROC) curve analysis. These results were further verified in the data set GSE126848 and a qRT-PCR analysis. The presumed abundance of immune cells was analyzed by the CIBERSORT algorithm. Histological analysis was used to validate these results. Correlation analysis of hub genes and immune cells was performed by “Spearman” rank correlation analysis. 

Key findings: We obtained 26 ferroptosis-related and 27 autophagy-related DEGs by the intersection with genes related to ferroptosis and autophagy respectively. These 13 hub genes, IL1B, IL6, ATG5, ERN1, MYC, FOXO1, CCL2, VEGFA, ERBB2, CDKN1A, FOS, CXCR4, and MTOR, were obtained through the STRING website and Cytoscape software. Ferroptosis-related DEGs were involved in the production of molecular of immune response, the cellular response to chemical stress, the cellular response to external stimulus, the response to T cell activation, and the regulation of production of molecular mediator of immune response. Autophagy-related DEGs were involved in the process utilizing the autophagic mechanism, the cellular response to external stimulus, and the cellular responses to stress. In addition, these 13 hub genes had a high accuracy in predicting the outcome of healthy control (HC) and NAFLD. Also, mast cells activated, monocytes, macrophages M1, T cells gamma delta, macrophages M2, plasma cells, eosinophils, and dendritic cells resting closely related to these hub genes. 

Significance: IL1B, IL6, ATG5, ERN1, MYC, FOXO1, CCL2, VEGFA, ERBB2, CDKN1A, FOS, CXCR4, and MTOR was identified as new diagnostic biomarkers for NAFLD. In addition, immune cell infiltration may be involved in the occurrence and development of NAFLD.

Note:
Funding declaration: This study was supported by the Science and Technology Research Project of the Education Department of Jiangxi Province (Grant GJJ190913) and Jiangxi Provincial Health Commission Science and Technology (Grant 20192654).

Conflict of Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Ethical Approval: All animal experiments were performed following the guidelines of the Animal Experiment Administration Committee of Fourth Military Medical University.

Keywords: Ferroptosis, autophagy, immune infiltration, Non-alcoholic fatty liver disease, Bioinformatics analysis, diagnostic biomarkers