Discussion
In recent years, different investigations in the area of multi-center genomics research from gene to systems level and next-generation sequencing, clarified the various mechanisms specifically involved in the tumor progression [
29]. This study used bioinformatics analysis to further investigate the genes involved in GC pathogenesis. In addition, the current study tries to shed light on changes in disease progression and comorbidities related to age.
Eleven hub genes were identified in this study, most of which were involved in mitochondrial functions. Recently, many cancer investigations have focused on the mitochondria for further elucidation of the molecular mechanisms of carcinogenesis [
23]. Mitochondria have complicated and diverse functions, including cell signaling pathways involved in the oncogenesis, natural immunity, and preparation of building blocks for new cells, modulation of biosynthetic metabolism, cell death, control of the redox homeostasis and energy supply [
30‐
32]. Different studies provided evidence about the potential correlation between the protein alterations and the number of mitochondria, as well as abnormal components and function of the mitochondria with several human cancer progression and prognosis in patients, signifying the possible functionality of mitochondria in new anti-tumor therapeutic strategies by targeting the mitochondrial proteins or metabolism [
29]. However, the characteristics of the respiratory and metabolic changes in GC have not still been fully elucidated.
Although the observation of mitochondrial dysfunction in various cancers, the concept of the involvement of both mitochondrial glycolysis and metabolism in cancer cells is controversial. On the other hand, the critical role of the mitochondria in the maintenance of cancer is inevitable. Consequently, there have been several discussions and investigations about the alterations in the function of the mitochondria and their protein expression in various human cancers [
23]. Based on Warburg’s observations, he suggested that the aerobic use of glucose in cancerous cells shows mitochondrial respiration impairment which might be the inherent cause of cancer [
23]. Today, it has been demonstrated that genetic and molecular complications lead to an irregular proliferation of the cancer cells, which might change their biochemical metabolism, including enhancement of aerobic glycolysis, usually without any corruption in the function of the mitochondria [
23,
33].
Mitochondrial quality control and biogenesis are enhanced in tumors. In addition, mutations in the enzymes of the nuclear-encoded mitochondrial tricarboxylic acid (TCA) cycle have been reported in some tumors leading to the production of oncogenic metabolites. However, negative selection is exploited for pathogenic mutations of the mitochondrial genome [
23]. It has been reported that removal of the mtDNA leads to the restriction of tumorigenesis, and human cancers with mutant mitochondrial genomes are shown to be quite benign. Therefore, mitochondria have a multifunctional and critical role in the pathogenesis of cancers, and mitochondria seem to be a promising therapeutic target for cancer [
23,
33,
34].
The respiratory chain, located in the mitochondrial membrane, is composed of 5 complexes, including Complex I or CxI (NADH dehydrogenase), Complex II (SDH), Complex III (cytochrome bc1), Complex IV (cytochrome c oxidase), and Complex V (ATP synthase) [
23]. In our study, most of the identified hub genes are related to complexes I, IV and V. Among these genes, ATP5A1, ATP5B, ATP5D, and MT-ATP8 encode different subunits of the ATP synthase. “F1F0 ATP synthase”, also known as “mitochondrial membrane ATP synthase”, recruits a proton gradient across the inner membrane, which is generated by electron transport complexes of the respiratory chain to produce ATP from ADP. [
35]. Liu et al
. introduced ATP5A1 as one of the up-regulated genes in GC [
36]. ATP5B is one of the most important subunits of ATP synthase and increases cellular ATP levels. Wang et al
. found that high ATP5B expression in tumor tissues of GC is positively correlated with age, tumor size, the TNM stage, lymph node metastasis, and patients’ poor prognosis. They reported that ATP5B overexpression in GC cells caused ATP-promoting migration, invasion, and proliferation. In their study, an increase in MMP2 expression results in the phosphorylation of FAK, and phosphorylated AKT was observed by ATP5B overexpression in GC cells. Also, an increased level of extracellular ATP happens after ATP5B overexpression through the intracellular ATP secretion, and the FAK/AKT/MMP2 pathway is activated. Activation of the ATP5B-induced downstream pathway is activated through the P2X7 receptor. Inhibition of P2X7, FAK, AKT, and MMP2 results in the suppression of proliferation, migration, and invasion of GC cells. In conclusion, studies by Wang et al
. showed that ATP5B involves in GC tumor progression through FAK/AKT/MMP2 pathway [
37]. Therefore, ATP5B may serve as a poor prognosis biomarker and a beneficial therapeutic target for GC.
MT-ATP8, as one of the components involved in the OXPHOS pathway, is a mitochondrial gene encoding membrane subunit 8 of ATP synthase. Several genetic syndromes that contributed to mitochondrial dysfunction depicted by a reduced OXPHOS ability have been explained [
38]. In addition, reduced OXPHOS in the mitochondria has been reported in many types of cancer cells which is related to either or both reduced flux in the TCA cycle and/or respiration [
30]. Furthermore, MT-ATP8 was reported to have mutations in GC [
39]. In the current study, we reported the genes related to the OXPHOS pathway, however, the OXPHOS reduction cannot be demonstrated based on our data, and it needs to be further investigated.
ATP5D encodes the subunit delta of the catalytic core of ATP synthase, F1. Wei et al
. found that overexpression of PSMB10, VPS13D, NDUFS8, ATP5D, POLR2E, and HADH were correlated with adverse overall survival in acute myeloid leukemia (AML) [
40].
NDUFS3, MT-ND4, and MT-ND6, as three other hub genes identified in this study, encode subunits of CxI. MT-ND6 encodes NADH dehydrogenase 6, part of CxI in the mitochondria. CxI is involved in the first step in the electron transport process, in which the electrons are transferred from NADH to ubiquinone. Electrons are subsequently thrown from ubiquinone through various other enzyme complexes leading to the preparation of energy for ATP generation [
41].
Ishikawa et al
. showed that the MT-ND6 mutation resulted in the suppression of CxI activity and induction of ROS production, leading to the stimulation of metastasis in breast and lung cancer cells [
11]. There is one report for MT-ND6 in GC which is based on bioinformatics analysis [
42].
MT-ND4 encodes NADH dehydrogenase 4, a part of CxI, one of the enzyme complexes involved in OXPHOS. In the whole mitochondrial genome sequencing study in GC, it has been reported that the tumor has remarkably more variants in the MT-ND4 region [
39].
NDUFS3 encodes one of the iron-sulfur protein components of mitochondrial NADH: ubiquinone oxidoreductase (CxI). Recent studies in GC showed the prognostic capabilities of Nicotinamide N-methyltransferase (NNMT). They found that NNMT expression was positively connected to clinical pathologic stage, tumor size, and lymph node status in GC. Silencing the NNMT gene resulted in the inhibition of proliferation, invasion, and migration of the GC cells. These results represent the potential of NNMT as a beneficial prognostic marker of GC. It has been observed in neuroblastoma that the presence of NNMT could meaningfully reduce the death of SH-SY5Y cells, and the effects of NNMT were positively correlated with the elevated intracellular ATP content, ATP/ADP ratio, and CxI activity, as well as a decrease in the degradation of the NDUFS3 subunit of CxI [
43]. There are various studies on the evaluation of the role of NDUFS3 as a part of CxI in different cancers. NDUFS3 has been suggested as a biomarker for the breast. ovarian and a few kidney cancers. However, there is no report on the evaluation of the role of NDUFS3 in gastric cancer [
44].
Among the identified genes in this study, COX7A2 and COX6C, encode cytochrome c oxidase subunits. COX7A2 encodes subunit 7A2 of the cytochrome c oxidase, which is the terminal module of the respiratory chain in the mitochondrial and catalyzes the transfer of electrons from the reduced cytochrome c to oxygen. Data extracted from MALDI-MSI experiments have represented that COX7A2 expression is correlated with the survival curve in GC [
45]. Elsner et al
. showed that increased expression of COX7A2 is a characteristic feature of intestinal metaplasia in the esophagus [
46].
COX6C encodes subunit 6c of the COX complex, which catalyzes the final step of the electron transfer chain. Recently, many investigations have reported the unusual level of COX6C in different cancerous and non-cancerous disease conditions such as diabetes, uterine leiomyoma, prostate cancer, melanoma tissues, breast cancer, and follicular thyroid cancer. It has been reported that the overexpression of NDUFA4 leads to substantial upregulation of the COX6C, which subsequently promotes GC cell proliferation and reduces apoptosis in these cells [
47].
RPL8 and RPS16 were also identified in the present study. RPL8 encodes ribosomal L8 protein, which is one of the components of the 60S subunit. Based on a bioinformatics analysis-based study, among the genes highly expressed in one of the clusters, few were associated with ribosomal protein-encoding genes such as RPL8 [
22]. RPS16 encodes ribosomal protein S16. The diseases associated with RPS16 include Diamond-Blackfan Anemia and Descending Colon Cancer [
23].
The oxidoreductase activity which was considerably enriched in our GO analysis has been investigated in various cancers, including the breast, liver, gastrointestinal tract, and kidney. Also, it has been shown that Xanthine oxidoreductase (XOR) expression is negatively correlated with poor prognosis in these malignancies [
48].
The OXPHOS pathway which was considerably enriched in our KEGG and cluster analyses produces energy in most cells, and as the final stage of cellular respiration, is composed of two parts: the electron transport chain (ETC) and chemiosmosis. In the ETC, the electrons are transferred from one molecule to the other, and the released energy from this transfer is applied in the formation of an electrochemical gradient. In chemiosmosis, ATP is made from the stored energy in the gradient. ATP synthase exploits the produced gradient of the proton to generate ATP after the phosphorylation of ADP. At the end of the ETC, oxygen accepts the electrons and makes water with protons [
30]. Four genes identified in the current study (ATP5A1, ATP5B, ATP5D, and MT-ATP8) encode the subunits of the ATP synthase, three genes (NDUFS3, MT-ND4, and MT-ND6) encode subunits of complex I, and two genes (COX7A2 and COX6C) encode subunits of complex IV. These findings highlight the role of mitochondrial respiratory chain components and OXPHOS pathway in GC pathogenesis.
Recent studies presented that expression levels of several genes, such as NDUFB7, UQCRC2, and UQCRQ in the OXPHOS pathway, were meaningly down-regulated in GC. In addition, studies showed that the enhanced expression of the genes in this pathway, such as UQCRQ, NDUFB7, and UQCRC2, positively correlated with a better prognosis [
49]. It has been also reported that defection in UQCRQ, as a subunit of complex III, can cause mitochondrial dysfunction, which is correlated with the pathogenesis of ulcerative colitis [
23]. Also, it has been recently reported that a mutation in the UQCRC2 gene might lead to a deficiency of the mitochondrial complex III, a relatively rare disease [
50]. Substantially lower content of UQCRC2 has also been reported in breast cancer cells compared to normal cells [
51]. Downregulation of this gene has also been reported in glioma and GC [
52]. According to these various investigations [
23] showing the role of mitochondria in cancer, the malfunctioned bioenergetic mitochondria can be suggested as a symbol of tumorigenesis.
It has been reported that besides the role of the oncogenes and tumor suppressors in the expression control of the metabolic enzymes, mitochondrial energy metabolism is also affected by environmental conditions, and it has been shown that mitochondrial dysfunction is one of the characteristics of tumor cells [
33]. Under various conditions, including mitochondrial DNA mutation or activation of the oncogenes, cancer cells have different abilities to use oxygen, and few studies are available on the identification of the strict relationship between the metabolic changes and composition and activity of mitochondrial complexes [
30]. A substantial decrease in the activity of NADH dehydrogenase and complex I protein content has been reported in renal and lung cancer, and a reduction in the activity of complex I was also reported in thyroid cancer which was related to ND1 gene [
30].
Association of mitochondrial dysfunctions with OXPHOS complexes content was also reported in a few studies [
33]. A decrease in the ATP synthase content has been reported in renal cancers and chromophilic tumors, indicating inefficient structure and activity of the complex in mitochondria [
53]. It has been suggested that low amounts of ATP synthase may have a critical role in the metabolism of cancer cells because carcinogenesis has been observed to have substantial effects on the expression of the F1-ATPase beta subunit leading to the changes in the control mechanisms of mitochondrial differentiation [
54].
The switching of cellular energy production from OXPHOS by mitochondria to aerobic glycolysis, named the Warburg effect, happens in several cancer types [
33]. One of the major characteristics of fast-growing cancer cells is to maintain an increased glycolysis level providing enough ATP irrespective of the existence of oxygen in the environment [
49,
55]. However, the importance of this switching for GC development is poorly understood. Recently,
Feichtinger et al
. investigated the expression of OXPHOS complexes in GC using immunohistochemistry. They found that CxI expression was significantly reduced in the intestinal type of GC (but not the diffuse type of GC). Higher expression of complex I and II was seen in larger tumors, and higher expression of complex II and III was observed in higher grades [
56]. On the other hand, Su et al
. found that all of the identified DEGs involved in OXPHOS were down-regulated. They also reported that GC has substantially changed metabolic processes, including NADH dehydrogenase complex assembly and tricarboxylic acid cycle, consistent with our KEGG analysis results [
49]. Therefore, the expression status of the OXPHOS complexes in GC needs to be further studied to elucidate the exact role of these complexes in the pathogenesis of GC.
Mitochondrial changes have been also reported to be correlated with tumor cell migration, invasion, and resistance to chemotherapy [
23]. The primary suggestion that mtDNA and mitochondrial dysfunction might be involved in metastasis originated from Ishikawa et al., who reported that mitochondria transfer from more aggressive tumors into less aggressive ones led to enhanced aggressiveness [
57]. Furthermore, various mutations in the components of the ETC complex I resulted in enhanced ROS levels, causing an enhanced metastatic characteristic [
57,
58]. Particularly, mutations of ND4 and ND5 in breast cancer cell line [
59] and ND6 in lung cancer cell line [
41] have been observed to cause enhanced metastasis. Other mtDNA SNPs also changed the efficiency of metastasis [
60]. In addition to serving as metabolic ‘powerhouses of the cell’, mitochondria have co-evolved with their hosts to serve as critical signaling hubs in several pathways. For example, mitochondrial signaling can influence cancer and metastasis in inflammation [
61] and apoptosis [
62]. Most of the identified genes in our study (ATP5A1, ATP5B, ATP5D, MT-ATP8, COX7A2, COX6C, ND4, ND6, NDUFS3 and RPS16) contribute to mitochondrial function suggesting their potential involvement in GC pathogenesis through various pathways. However, the correlation between these genes in regulating mitochondrial function and cancer pathogenesis continues to be unclear and requires to be validated by more research.
Many of the enriched pathways in our KEGG analysis were also related to neurodegenerative diseases and based on the previous studies [
27], Sirutin 3 (SIRT3) is one of the common genes among these pathways. SIRT3 is a member of the sirtuin protein family known as class III histone deacetylases. Recent investigations have focused on SIRT3 due to its function in stress resistance, aging, neurodegenerative disease, and cancer. SIRT3 manages energy requests in situations, including mitochondrial metabolism. The elimination of reactive oxygen species and prevention of cancer cell development or apoptosis are the abilities of SIRT3, which highlights its critical role in cancer and various diseases, including Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson’s disease, and Huntington's disease [
27].
The results of pathway enrichment analysis in our study also showed that the metabolic processes are the most affected in GC. Recent investigations have focused on the elucidation of the correlation between metabolic reprogramming and the pathogenesis of cancer. Specifically, regulation of the metabolism and cancer investigation is more brought into intense attention with the development of metabolomics [
63]. Metabolomics offers comprehensive knowledge of the metabolic profiles of certain cancers. It may provide an excellent tool to find biomarkers for prognosis, diagnosis, metastatic surveillance, and therapeutic sensitivity estimate. Various metabolic changes have been identified in GC, including glucose metabolism, amino acid metabolism, lipid metabolism, and nucleotide metabolism. In addition to the mentioned metabolic alterations in GC, changed levels of other metabolites, including creatinine and inositol, have also been reported in GC [
63].
Our results also showed the potential significance of RNAs in GC pathogenesis, confirming recent studies focused on elucidating the role of RNAs in the pathogenesis of this cancer [
64]. Using an inclusive assessment of the results from other studies, it has been demonstrated that circular RNAs (circRNAs) regulate the cellular biological behaviors in GC, including epithelial-mesenchymal transition (EMT), proliferation, migration, and invasion. In addition, circRNAs are correlated to the GC characteristics, including tumor stage, differentiation of the tumor, and metastasis. Therefore, circRNAs might be suitable to be used as prognostic or diagnostic biomarkers in GC. In addition, the circRNAs involved in GC pathogenesis can be used as targets in GC treatment [
64].
By integrated bioinformatics analysis in 2022, Zhang et al. studied the stomach adenocarcinoma and introduced seven hub genes (EWSR1, ESR1, CLTC, PCMT1, TP53, HUWE1, and HDAC1) as related genes to occurrence of stomach adenocarcinoma [
65]. Through a systems biology approach in 2022, Salarikia et al. reported that medications such as pantoprazole, omeprazole, imatinib, troglitazone, fostamatinib, and amiloride might be useful in GC treatment. They also found that disorders such as liver fibrosis, ovarian carcinoma, breast carcinoma, lung cancer, liver carcinoma, and prostate cancer might have correlation with gastric adenocarcinoma through certain genes, including
mt2a, fn1, hgf, col1a1, col1a2, and
mmp2. In their study, overexpression of signaling pathways, including extracellular matrix organization, cell division, and cell cycle, as well as downregulation of ion transport and digestion pathways were also indicated. They also reported that their identified hub genes in gastric adenocarcinoma participated in platelet activation, focal adhesion, gastric acid secretion, cell cycle and HPV infection pathways [
66]. In 2018, Saberi Anvar et al. used a systems biology approach for GC biomarkers and found that cell cycle, neutropin signaling pathway and nucleotide excision are the most enriched signals. Also, they reported TP53, HNF4A and TAF1 as the most important nodes in the PPI network [
67].
We also found three genes that present significant changes in the elder population: MTRNR2L12, EIF4G2, and EIF4B. MTRNR2L12 has no annotations available, so we suggest further analyzing this possible target. EIF4G2, as reported in the STRING database appears to play a role in the switch from cap-dependent to IRES-mediated translation during mitosis, apoptosis and viral infection [
68]. EIF4B acts downstream of mTOR signaling in response to growth factors and nutrients to promote cell proliferation, cell growth and cell cycle progression [
69]. In addition, it regulates protein synthesis through phosphorylation of EIF4B, RPS6 and EEF2K, and contributes to cell survival by repressing the pro-apoptotic function of BAD [
69]. EIF4G2 (eukaryotic initiation factor 4G2) and EIF4B (eukaryotic initiation factor 4B) are both annotated as aging genes. The downregulation of eIF4B impacts the overall protein translation and the aberrant which is strongly associated with cancerous outgrowth. Moreover, the altered expression of EIF4G2, as reported in some recent work [
70] is related to poor prognosis of GC. Therefore, our analysis may suggest that the expression changes of this gene with age may increase the risk of poor prognosis and suggest further investigation.
GEPIA (Gene Expression Profiling Interactive Analysis) web server is an important resource for the analysis of gene expression based on cancerous and normal samples from the GTEx and the TCGA databases and GEPIA2 is an improved version [
71]. The Kaplan–Meier overall survival curves of GC patients extracted from GEPIA2 showed that high expression of the eleven hub genes identified in this study is associated with decreased overall survival rate [
71]. The survival rate after 100 months for GC patients with overexpression of ATP5A1, ATP5B, ATP5D, COX6C, COX7A2, MT-ATP8, ND4, ND6, NDUFS3, RPL8, and RPS16 genes showed < 20%, 40%, > 20%, 40%, > 20%, < 40%, 30%, > 30%, > 20%, > 20%, and > 20%, respectively [
71]. Accordingly, ATP5A1 overexpression is associated with the lowest survival rate in GC patients. Therefore, inhibition of ATP5A1 is suggested to be further studied as a therapeutic tool for survival improvement in GC patients that show overexpression of this gene.Although our study suggested the probable relevance between our identified genes/CREs and GC pathogenesis, little is known about the relevance of these genes/CREs to GC. The identified genes in this study might be promising diagnostic and prognostic biomarkers for GC. Moreover, clinical validation and long-term follow-up data for GC patients with a large sample size are required for investigating these genes as prognostic or diagnostic biomarkers. If these genes' diagnostic/prognostic importance is validated, they could be introduced as factors to define the treatment modality for GC. Research on targeted changes in the expression of these genes can offer novel therapeutic modalities.