Identification of key genes and pathways in mild and severe nonalcoholic fatty liver disease by integrative analysis

Abstract

Background: The global prevalence of nonalcoholic fatty liver disease (NAFLD) is increasing. The pathogenesis of NAFLD is multifaceted, and the underlying mechanisms are elusive. We conducted data mining analysis to gain a better insight into the disease and to identify the hub genes associated with the progression of NAFLD.

Methods: The dataset GSE49541, containing the profile of 40 samples representing mild stages of NAFLD and 32 samples representing advanced stages of NAFLD, was acquired from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) were identified using the R programming language. The Database for Annotation, Visualization and Integrated Discovery (DAVID) online tool and Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database were used to perform the enrichment analysis and construct protein-protein interaction (PPI) networks, respectively. Subsequently, transcription factor networks and key modules were identified. The hub genes were validated in a mice model of high fat diet (HFD)-induced NAFLD and in cultured HepG2 cells by real-time quantitative PCR.

Results: Based on the GSE49541 dataset, 57 DEGs were selected and enriched in chemokine activity and cellular component, including the extracellular region. Twelve transcription factors associated with DEGs were indicated from PPI analysis. Upregulated expression of five hub genes (SOX9, CCL20, CXCL1, CD24, and CHST4), which were identified from the dataset, was also observed in the livers of HFD-induced NAFLD mice and in HepG2 cells exposed to palmitic acid or advanced glycation end products.

Conclusion: The hub genes SOX9, CCL20, CXCL1, CD24, and CHST4 are involved in the aggravation of NAFLD. Our results offer new insights into the underlying mechanism of NAFLD progression.

Keywords: Computational biology; Fatty liver; Nonalcoholic fatty liver disease.

Fig. 1

Heat map (A) and volcano map (B) of the identified differentially expressed genes (DEGs) between mild (n = 40) and advanced (n = 32) NAFLD livers based on the GSE49541 dataset.

Fig. 2

Top 11 pathways and biological functions enriched in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and Gene Ontology (GO) analysis related to DEGs.

Fig. 3

Protein–protein interaction (PPI) and module analyses. (A) PPI network and module analyses of DEGs in GSE49541 dataset. (B–C) Significant modules, module 1 (B) and module 2 (C), selected from PPI network analysis. The color and size of node are relative to its degree (darker the color and larger the size, the greater is the degree). The strength of the confidence score is symbolized by the thickness of the line (the thicker the line, the higher is the confidence score).

Fig. 4

Transcription factor target networks in the top 3 modules using the iRegulon plugin. Blue octagon nodes represent the predicted transcription factors. Pink oval nodes represent the transcription factor-regulated genes.

Fig. 5

Validation of the potential key genes in the livers of NAFLD mice and in cultured HepG2 cells exposed to glucolipotoxicity. C57BL/6N mice were fed a high fat diet (HFD) for 18 (n = 6) and 24 (n = 3) weeks. Age-matched C57BL/6N mice fed a normal diet (n = 3) were used as the control. (A) Oil red O staining of liver tissues. Scale bar = 50 μm. (B) The percentage of total adipose tissue (as detected by magnetic resonance imaging scan) to body weight. (C) Relative mRNA levels of hub genes in mouse liver tissues detected by real-time quantitative PCR (qPCR). (D) Relative mRNA levels of hub genes determined by qPCR in HepG2 cells cultured with palmitic acid (PA) or vehicle for 24 h (n = 3). (E) Relative mRNA levels of hub genes detected by qPCR in HepG2 cells cultured with advanced glycation end products (AGEs) or vehicle for 24 h (n = 3). Data are expressed as the means ± standard deviation. Statistical analysis was conducted using one-way ANOVA followed by the post-hoc Tukey–Kramer test. ^aP < 0.05 (vs. control). ^bP < 0.05 (vs.18-week HFD exposure).