Identification of key target genes and pathway analysis in nonalcoholic fatty liver disease via integrated bioinformatics analysis

Nonalcoholic fatty liver disease (NAFLD) is the most common type of chronic liver disease with a prevalence rate of 25% worldwide (1). Several lifestyle-related factors are associated with incident fatty liver such as alcohol intake, lower physical activity, smoking, and shift work. Poor lifestyle choices are often the main cause of fatty liver, these include smoking, drinking, lack of physical activity, and shift work, etc. In addition, high triglycerides, type 2 diabetes mellitus, obesity, and hypertension are associated with incident fatty liver. Therefore, lifestyle modification is strongly recommended to prevent fatty liver (2, 3). It is difficult to detect this problem in the earlier stages of the disease, and may thus further develop into advanced liver diseases, such as cirrhosis and hepatocellular carcinoma, bringing forth clinical challenges to the treatment of NAFLD (4). In the literature, the severity of NAFLD in patients with type 2 diabetes and obesity will be significantly affected, increasing the degree of deterioration of liver fibrosis and the possibility of further development of end-stage liver disease (5, 6, 7). Likewise, studies have shown that when NAFLD patients suffer from cardiovascular diseases and dyslipidemia, these factors have a negative impact on the natural progression of NAFLD (8, 9, 10). Nearly 40% of patients with NAFLD die of complications, as previously reported (1). However, the detailed mechanisms under which NAFLD develops remain largely unknown. Diet adjustment and weight loss can improve NAFLD, but it is difficult to maintain. Moreover, the theory of insulin resistance has been widely accepted clinically. Insulin sensitizers have a certain therapeutic effect, but they can cause adverse reactions such as increased body weight and its therapeutic target is too limited. Therefore, this study aimed at finding new molecular targets to provide a theoretical basis for new and effective treatment methods of NAFLD.

Long non-coding RNA (lncRNA) is the main component of the human transcriptome. Long non-coding RNA plays an important role in regulating cell migration, proliferation, invasion, and metastasis. It can also be used as a diagnostic marker or therapeutic target for malignant tumors and other diseases. Competitive endogenous RNA (ceRNA) is a transcript with the same microRNA (miRNA) response element, which binds to miRNA to compete and regulate its target gene, thereby affecting the biological behavior of the disease. Studies have confirmed that the mutual regulation between lncRNA and miRNA and their downstream target genes plays an important role in the occurrence and development of diseases (11).

The inflammatory component of nonalcoholic steatohepatitis (NASH) is more difficult to capture with ultrasound-assisted techniques. Although more and more technologies are applied in clinical practice, such as quantitative and contrast-enhanced ultrasound, there are still many technical barriers to be broken; and not all technologies have been successful in clinical and research practice (12). Due to the limitations of liver biopsy, searching for non-invasive and reliable diagnostic biomarkers for NAFLD is a priority for current research. Bioinformatics has been widely used to explore biomarkers of different diseases, but NAFLD-related biomarkers need to be further explored to help the early diagnosis and prognosis evaluation of NAFLD (13).

In this study, human samples from the Gene Expression Omnibus (GEO) database were used to identify key genes related to NAFLD and non-NASH samples during the baseline and 1-year follow-up time point, and to explore the underlying mechanism of NAFLD and develop new NAFLD diagnostic biomarkers. Then, the lncRNA–miRNA–mRNA network related to NAFLD was constructed by mapping the differentially expressed RNAs (DERs) into a global triple network via starBase and miRcode databases. This was done to identify which RNAs can be used as sensitive and specific markers for NAFLD. Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to explore the potential regulatory functions of RNAs. Finally, the PharmGKB database was used to search and obtain gene-related drug molecules in the ceRNA regulatory network and then build a gene–drug connection network to screen out important gene molecules and KEGG signaling pathways involved in genes.

The characteristics of NAFLD include necrotizing inflammation and lipid accumulation in the liver, as well as continuous improvement of living standards leading to over-nutrition. In addition, bad living habits lead to the incidence of NAFLD on a global scale (27). The specific mechanism of the transition from benign steatosis to steatohepatitis in NAFLD is not fully understood, and there are currently no pharmacological options for the treatment of NAFLD. Therefore, the treatment of NASH mainly depends on lifestyle changes, such as strengthening exercises, reducing weight, and a light diet (28). Although current studies have shown that weight loss improves the histological characteristics of NAFLD, most patients have not however achieved the goal of curing NAFLD. There are some potentially valuable molecules, nevertheless, which are currently being clinically evaluated (29). For example, PNPLA3 (30), TM6SF2 (31), MBOAT7 (32), and HSD17B13 (33), molecules that predispose an individual to the spectrum of NAFLD-related disease, have been found to play a role in macrophage phagocytosis, immune response, oxidative stress, and inflammation, insulin signaling, and lipid metabolism in NAFLD susceptibility and progression (34). But there is no unmet clinical need for drug discovery and development for patients with NAFLD. Increased levels of toxic lipids (free fatty acids or free cholesterol) can lead to liver cell damage and trigger inflammation is the pathogenesis of NAFLD as is currently understood. In addition, oxidative stress, pro-inflammatory chemokines and cytokines have been proven to lead to liver inflammation, which, in turn, leads to damage and fibrosis of the liver. Therefore, the identification of pro-inflammatory cytokines related to lipotoxicity may improve our understanding of the pathogenesis of NAFLD, helping to develop new pharmacological methods.

In this study, a total of 220 overlapping DERs were identified between the baseline and 1-year follow-up time points. In addition, functional enrichment analysis of overlapping DERs, based on online DAVID analyses, revealed 22 significantly related GO biological processes and 9 KEGG pathways, with P < .05 as the cutoff criteria. We found that chemotaxis (P = 3.110E-04), unsaturated fatty acid biosynthetic process (P = 4.770E-04), and cell-cell signaling (P = 1.513E-03) were the three most significant pathways in GO biological processes. Meanwhile, fatty acid metabolism (P = 2.300E-04), PPAR signaling pathway (P = 1.090E-03), and Toll-like receptor signaling pathway (P = 1.479E-03) were the three most significant pathways in KEGG signaling pathways. Afterwards, a ceRNA regulatory network was constructed. The GO and pathway enrichment analyses indicated that the mRNAs of the ceRNA regulatory network were involved in various important biological functions and metabolic pathways associated with NAFLD, including lipid biosynthetic process, steroid metabolic process, steroid biosynthetic process, biosynthesis of unsaturated fatty acids, terpenoid backbone biosynthesis, heparan sulfate biosynthesis, Cytokine–cytokine receptor interaction, Insulin signaling pathway, and the pathways in cancer. To further understand the functional mechanism of the ceRNA network, a drug regulation gene network was constructed which included 154 gene–drug connection pairs. Subsequently, LEPR, CXCL10, and FOXO1 were investigated using the PharmGKB database. It was revealed that the therapeutic effect of antipsychotics, atorvastatin, valproic acid, risperidone, clozapine, olanzapine, simvastatin, and quetiapine were produced, thus possibly targeting to LEPR through the Cytokine–cytokine receptor interaction pathway. The therapeutic effect of Peginterferon alfa-2a and peginterferon alfa-2b were produced by targeting CXCL10 through the Cytokine–cytokine receptor interaction pathway. The therapeutic effect of Epirubicin, cyclophosphamide, and fluorouracil were produced by targeting FOXO1 through the Insulin signaling pathway or the pathways in cancer.

LEPR is responsible for encoding the leptin receptor that binds to leptin in target tissues. Due to its role in regulating lipid metabolism and insulin resistance, it is considered to be a candidate gene for NAFLD and coronary atherosclerosis (35). Simultaneously, An et al. (36) found that LEPR Q223R polymorphism may lead to a significant risk of NAFLD and coronary atherosclerosis, which is consistent with the results of this study. The CXC motif chemokine ligand 10 (CXCL10) is a particularly important pro-inflammatory cytokine related to lipotoxicity, which can recruit inflammatory cells to the site of tissue injury (37, 38). Studies have shown that CXCL10 is upregulated in NAFLD patients (39), and revealed that CXCL10 may be a key molecule that contributes to the transition from benign steatosis to steatohepatitis, promoting liver cell damage and inflammation (40).

Our study revealed that peginterferon alfa-2a and peginterferon alfa-2b can downregulate the expression of CXCL10, suggesting a potential role of CXCL10 in the development of intrahepatic inflammation through the Cytokine–cytokine receptor interaction pathway, and demonstrated that CXCL10 is an independent risk factor for patients with NAFLD. FOXO1 is an important transcriptional effector. It is widely expressed in various types of tissues and plays an important role in the signaling pathway of insulin and insulin-like growth factor 1 (41). In addition, the expression levels of most genes related to adipocyte differentiation are affected by the coordination of FOXO1 (42). Yue Li et al. (43) conducted a comprehensive analysis of the relevant information about the activity of FOXO1 in lipid metabolism, and found that FOXO1 has a significant inhibitory effect on the production of fibrotic effector cells, and pointed out that FOXO1 has the potential to become a target for the treatment of NAFLD, but the related mechanism needs to be further verified by experiments (44). L. Valenti et al. (45) found that FOXO1 may affect the susceptibility of NAFLD, and regulating the level of FOXO1 mRNA in order to regulate the relevant cytokines in insulin signaling to promote the progression of liver injury. This study found that epirubicin, cyclophosphamide, and fluorouracil can downregulate the expression of FOXO1, suggesting that these drugs may produce therapeutic effect by targeting FOXO1 through the insulin signaling pathway or the pathways in cancer. However, further study is necessary to validate this hypothesis.

In conclusion, this study constructed and analyzed a ceRNA network, a network which may provide some evidence for future studies focusing on the molecular mechanisms of NALFD. LEPR, CXCL10, and FOXO1 may function as ceRNAs to serve critical roles in NALFD. In addition, antipsychotics, atorvastatin, valproic acid, risperidone, clozapine, olanzapine, simvastatin, quetiapine, peginterferon alfa-2a, peginterferon alfa-2b, epirubicin, cyclophosphamide, and fluorouracil may produce therapeutic effect for patients with NALFD.