In the present study, PPI networks were produced based on differential gene expression provided from tissue liver in simple steatosis and NASH patients compared with healthy controls. Network analysis of PPI networks helped us detect common genes and pathways implicated in these diseases.
Nodes Analysis in the PPI network for DEGs of simple steatosis vs. healthy controls demonstrated that interleukin-6 (IL-6), and signal transducer and activator of transcription 3 (STAT3) and C-X-C Motif Chemokine Ligand 8 (CXCL8) had the top three highest degrees. Interleukin-6 is a family of cytokines synthesized and secreted by monocytes, macrophages, and neutrophils after stimulation of toll-like receptors (TLRs) by lipopolysaccharides (LPS). IL-6 is bounded into its receptor (IL-6R), then the IL-6/IL-6R complex was bounded to gp130, which activates STAT1 and STAT3. Subsequently, activated STAT3 up-regulates inhibitor of cytokine signaling . There are controversial reports regarding the level of IL-6, its receptors and the progression of liver injuries. For instance, Kroy et al. reported mice with IL-6 and gp130 knockout promoted hepatic steatosis . However, Hong and coworkers showed IL-6 reduced liver steatosis in NAFLD mice models and proposed remedial effects of IL-6 administration in the treatment of fatty liver patients . The second hub gene is STAT3. STATs are transcription factors present in the cytoplasm as inactive form (dephosphorylated STATs) that tyrosine kinases activate STATs. Phosphorylated STATs produce dimers; thus, dimerized STATs translocate to the nucleus and bind to target genes. Therefore, their effects including response to ILs and growth factors and regulation of cell growth, differentiation, and motility were performed . Studies demonstrate that STATs are dephosphorylated by T-Cell Protein Tyrosine Phosphatase (TCPTP). Since TCPTP was oxidized and inactivated in the obese mice model of NASH, it has been observed to increase STATs signaling . Besides, mice with TCPTP-deficient elevated phosphorylated STAT1 and STAT3 and correlated with worsening NASH . Consistent with these findings, one of the transcriptional targets of STAT-1 or STAT-3 is Cxcl9 that is up-regulated in NASH patients . The up-regulation of Cxcl9 gene expression in NAFLD patients accompanied by the exacerbation of fibrosis .
The top three genes in the PPI networks of NASH vs. healthy controls were IL-6, c-MYC (MYC), and JUN. MYC, a proto-oncogene, is a transcription factor that regulates several biological processes such as cell growth, proliferation, and apoptosis. Mice with transgenic c-myc expression showed increased levels of c-myc are correlated with ROS accumulation, p53 inhibition, and hepatic disease progression after feeding ethanol. Additionally, patients with alcoholic liver disease (ALD) showed up-regulation of c-myc expression that may lead to acyl-CoA oxidase (AKT) activation and p53 inhibition . Another study reported that enhanced protein expression of MYC accompanying elevated level of copper contributes to the development of cirrhosis to hepatocellular carcinoma (HCC) . SIRT7 is a kind of decarboxylases that inhibit endoplasmic reticulum (ER) stress, accumulation of unfolded proteins via stabilizing at the ribosomal proteins promoters, and interacting with MYC to suppress some gene expression. Mice with SIRT7 -deficient progress to fatty liver disease; subsequently, MYC's inactivation prevents the progression of fatty liver disease because of SIRT7 deficiency . Another hub gene was JUN, a proto-oncogene that is an eminent member of the activator protein 1 (AP1) family. The expression of c-Jun elevated in liver biopsy of simple steatosis and NASH compared with healthy controls, and hepatic expression of c-Jun increased in the NASH model of mice [24, 25]. Besides, c-Jun diminished hepatic expression of the type II inositol 1,4,5-trisphosphate receptor (ITPR2), a calcium channel leading to liver regeneration . Another study showed that c-Jun expression could progress simple steatosis and NASH by regulating Osteopontin's expression . The AP-1 transcription factor c-Jun is a putative β-Catenin target gene and promotes hepatocyte survival, proliferation, and liver tumorigenesis, suggesting that c-Jun may be a key target β-Catenin signaling in the liver. c-Jun is also frequently expressed in human HCCs and acts as an oncogene in the liver .
The most degree three genes in the PPI networks of NASH vs. simple steatosis were ribosomal protein S27a (RPS27a), STAT3, and fibronectin1 (FN1). RPS27a is one ribosomal protein synthesized as a fusion protein (at the C terminus) along with ubiquitin (Ub) at the N terminus. This precursor protein quickly hydrolyzed to an Ub and RPS27a protein [28, 29]. There is evidence reporting ribosomal proteins related to apoptosis, which is one of NAFLD features and can promote the conversion of simple steatosis to NASH [30, 31]. Moreover, the expression of other ribosomal proteins, including RPL36A and RPL14, were down-regulated in obese, steatosis, and NASH patients compared with healthy controls . The down-regulated level of RPL36A and RPL14 can elevate apoptosis. FN is another hub gene that a glycoprotein presented in bio-fluids such as plasma and cell surface and the extracellular matrix implicated in cell adhesion and migration, tissue repair, and blood coagulation. A 5-year follow-up study from obese patients at an early stage of NASH has shown that parenchymal FN might be a marker for anticipating fibrosis progression . Furthermore, elevated FN levels from liver biopsy were associated with fibrosis progression in different liver diseases (alcoholic fatty liver, alcoholic hepatitis, alcoholic cirrhosis, chronic active hepatitis) compared with the control group .
The top five genes in the hepatic PPI networks of three data sets were STAT3, JUN, calnexin (CANX), FN1, and MYC. All these hub genes have been discussed above, except CANX. CANX is a transmembrane ER protein that is implicated in the folding of new glycoproteins . Growing evidence indicates that this chaperone is related to ER stress and apoptosis. The levels of CANX were reduced in liver tissue and its microsomes in NASH patients compared to healthy controls . Furthermore, some TFs relevant to ER stress enhanced, while some apoptosis markers reduced in liver NASH patients .
The shared genes among the three groups (SH, NH, and SN) for each dataset have been shown in Fig. 1a-c. There was no common gene for dataset GSE48452. However, dataset GSE63067 and dataset GSE89632 had two and three shared genes, respectively. These genes for GSE63067 were ICAM1 and PEG10. ICAM1 is a glycoprotein located in the cell surface and expressed in some cells such as liver, endothelial, epithelial hematopoietic cells. The Overexpression of ICAM1 may occur by some inflammatory cytokines, including IL-1 and TNF-α . The study of Ito et al. suggested that serum ICAM1 might be a diagnostic marker for NASH because its concentration increases in NASH patients compared with healthy individuals.
Moreover, there was a positive correlation between serum ICAM1 level and the severity of liver fibrosis and inflammation . Another study showed that hepatic expression of ICAM1 is significantly higher in NASH patients than in the simple steatosis and normal controls. A significant correlation was observed between the steatosis degree and hepatic ICAM1 expression . Another common gene, PEG10, which prohibits apoptosis via interacting with SIAH1, is a tumor suppressor protein. Overexpression of PEG10 is linked to some malignancies, such as HCC and chronic lymphocytic leukemia [39, 40]. Besides, PEG10 expression increased in simple steatosis and NASH patients, as well as its concentration is associated with severity of liver disease . Therefore, this protein might serve as promising therapies to prevent HCC in NAFLD patients. The shared genes among the three groups for dataset GSE89632 were FMO1, PEG10, and MT1A. Six FMO genes have been detected in mammalians that are important for the metabolism of various compounds, such as toxic and therapeutic substances . A prior study displayed that human liver FMO1 is expressed in the fetus, while FMO3 is the main isoform in adult . FMO3 and in smaller quantities, FMO1 metabolize trimethylamine (TMA) into trimethylamine- N -oxide (TMAO) . Methylamines (TMA and TMAO) are a type of metabolite produced by the gut microbiota through the degradation of choline into TMA and adsorbed into circulation and metabolized into TMAO in the liver . TMAO promotes inflammation and insulin resistance via elevating inflammatory cytokines.
Furthermore, TMAO inhibits bile acid production and impairs the main pathway to remove cholesterol from the body and causes TMAO-mediated atherosclerosis, and is associated with NAFLD's severity [44, 45]. Intestinal dysbiosis in cirrhotic patients is related to the low level of bile acids [46, 47]. Methylamines were known as markers of NAFLD, and metabolic syndrome and increased levels of TMAO are related to the severity of hepatic steatosis [48–50]. Another hub gene, MT1A, is a metalloproteins family with a high affinity for binding essential and toxic metals such as zinc and cadmium, respectively [51, 52]. MT1A's biological functions are protection against toxic metals, involvement in apoptosis, defense against oxidative stress (OS), proliferation and differentiation of cells, and protection against cytotoxicity as well as genotoxicity [53, 54, 52]. Additionally, MT1A has an indispensable role in the regeneration of hepatocytes . The down-regulation of MT1A gene expression has been demonstrated in simple steatosis versus healthy controls, and a decrease was observed in NASH patients compared with the control group . Also, it seems metallothioneins play a leading role in HCC pathogenesis, as down-regulated gene expression of MT1A isoform, MT1G, was observed in more than 60% of tumors relevant to the adjacent nonmalignant liver .
Venn diagrams (Fig. 3a-c) illustrate the number of shared DEGs that were found in the three datasets for SH, NH and NS groups. There are no shared DEGs for NS; however, one (PEG10) and two (IGFBP2 and PEG10) shared genes were observed in SH and NH groups, respectively. IGFBPs are a family of secreted proteins binding to insulin-like growth factors (IGFs), regulating IGFs bioavailability and its function . Recently evidence introduced this protein as a non-invasive biomarker for the development and pathogenesis of fatty liver disease [58, 59]. The IGFBP2 promoter was hypermethylated, and its mRNA expression downregulated in a mouse model of fatty liver; therefore, secretion of hepatic IGFBP2 and plasma level of IGFBP2 reduced . This observation is in line with human studies, as serum/plasma levels of IGFBP2 decreased in obese males and females and obese men with NAFLD and NASH compared with the non-obese control group [60, 58]. Reduced level of IGFBP2 is along with decreased oxidation of fatty acids and enhanced de novo lipogenesis in hepatocytes .
Nevertheless, IGFBP2 concentrations increased in fibrosis caused by chronic hepatitis C compared with healthy individuals. The levels of IGFBP2 were positively associated with fibrosis stages. This contradiction might be attributed to the participation of IGFBP2 in the modulation of the parenchymal structure in advanced liver fibrosis .
Shared genes among the three datasets were extracted after nodes analysis in the PPI network; these genes included CHI3L1, NRG1, FADS2, IGFBP2, and PEG10 (Fig. 2). CHI3L1, also called YKL40, belongs to a chitinase family member lacking chitinase activity and enriches in the liver. Some functions of CHI3L1 are the regulation of cell proliferation, differentiation, apoptosis, and cancer metastasis. It is also related to inflammation and infection . Many studies reported that CHI3L1 is a useful liver fibrosis marker in NAFLD and hepatitis B virus infection [63, 64, 62]. Liver fibrosis may be produced by accumulation and activation of liver macrophages . Besides, CHI3L1 suppresses hepatic macrophage apoptosis by inhibiting Fas expression and activation of phosphoinositide 3-kinase/protein kinase B signaling and increases hepatic fibrosis . Another gene was NRG1 that is a member of the epidermal growth factor (EGF) family and binds to a class of receptor tyrosine kinases, ErbB receptors. NRG1 is implicated in the differentiation, proliferation, and survival of cells . NRGs are local growth factors, while NRG1 has been found in plasma as a diagnostic marker for various maladies such as chronic heart failure (67) and acute lung injury associated with inflammation . NRG-1 treatment enhanced the fibrotic region in the liver from the animal model of liver fibrosis.
Furthermore, elevated expression of TGF- β, a mediator of fibrogenesis, was observed in isolated hepatocytes after NRG1 treatment compared with untreated cells. The fibrotic impact of NRG1 might be because of the phosphorylated ErbB3 receptor and activated the NRG-1/ErbB3 pathway . The other common gene was FADS2, the rate-limiting enzyme in the biosynthesis of unsaturated fatty acids and catalyzed linoleic conversion to γ-linolenic α‐linolenic acid stearidonic acid . Increased activity of delta-6 desaturase was observed in NASH patients and was related to DNA hypomethylation of two CpG sites in FADS2 . Also, Walle et al.  showed similar findings. They found a reduced level of liver DNA methylation in the FADS2 loci is associated with both more excellent serum activity of delta-6 desaturase and higher hepatic mRNA expression of FADS2. Furthermore, they suggested that FADS2 contributes to NAFLD's early stages and not in an advanced stage of hepatic fibrosis.