ITRAQ-based Bioinformatics Analysis Reveals the Potential Anticancer Effects of ATP4B in Gastric Cancer


 Background: Gastric cancer (GC) is one of the major malignancies of gastrointestinal tract. Hydrogen-potassium ATPase beta (ATP4B) gene is aberrantly downexpressed in gastric cancer that is associated with worse disease outcome. The objective of this study was to investigate the biological significance of ATP4B in GC carcinogenesis and development. Methods: The expression level of ATP4B was analyzed via clinical tissues and TCGA database. Then, we overexpressed ATP4B in SGC7901 and utilized isobaric Tags for Relative and Absolute Quantitation (iTRAQ) technique validate the ATP4B-regulated proteomics profile alterations. Bioinformatics analysis was performed to evaluate the biological processes of ATP4B in GC. Western blot was used for the verification of significant downstream proteins of ATP4B based on bioinformatics analysis data.Results: We identified 293 differentially expressed proteins between the ATP4B overexpressing and control groups in SGC7901, including 145 upregulated proteins and 148 downregulated proteins. GO enrichment analysis indicated that ATP4B-modulating downstream proteins were primarily related to mitochondria function and metabolism. ATP4B-induced enrichments of biological functions were partly associated with suppressing tumor advancement, illustrating an inhibitory role for ATP4B in the progression of GC. Co-expression interaction network analysis exhibited the significant alterations in p53 and STAT3/NF-κB signaling pathway. Consistently, KEGG pathway analysis showed that DEPs are enriched in cell metabolism and cancer-related signaling pathway. Western blot validated the activation of p53 pathway and the inhibition of NF-κB /CD44 pathway after ATP4B overexpressing in GC cells.Conclusion: ATP4B plays a critical anticancer effect by regulating p53/NF-κΒ/mitochondrial pathway. Our data suggested a novel role and mechanism for ATP4B in GC progression.


Introduction
As one of the most deadly threats to humans due to the high morbidity and mortality, gastric cancer (GC) is the third most frequent cause of tumor-related mortality in both sexes worldwide [1]. There were greater than 1,000,000 new cases and an estimation of 783,000 gastric cancer-associated mortalities globally in 2018 [2], which brought great socio-economic burden on patients, their families, and the whole society.
The advancement of diagnostic techniques and therapeutic strategies has been provided, but the prognosis of patients with this disease has not improved signi cantly. GC remains an important health burden worldwide owing to the lack of effective diagnostic biomarkers for early diagnosis [3]. Therefore, it is a critical need to discover the prospect novel diagnostic biomarker and new therapeutic targets for GC.
With the ongoing development of sequencing and proteomics mass spectrometry techniques, analyzing the alteration of gene expression pro les using high-throughput platforms provides more effective way to explore speci c biomarkers and potential targets for a wide range of cancers, including GC [4]. In recent years, several genes signi cantly associated with GC have been identi ed via bioinformatics analysis based on dynamic transcriptomics, such as COL2A1, ATP4B, ATP4A, COL11A1, EGFR, and GIF [5][6][7].
Among them, we pay a great attention to the biological function of ATP4B gene in GC which is aberrantly downregulated expression in gastric tumor tissues [6].
ATPase H + /K + transporting beta subunit (ATP4B) is mapped at human chromosome 13q34 [8], encoding a family of the P-type cation-transporting ATPases. ATP4B is mainly expressed in the parietal cells of the stomach and serves as a gastric function gene, which plays an essential role in the formation and secretion of hydrochloric acid [9,10]. GC is a long-term progressive disease from in ammation to atrophy, intestinal metaplasia, dysplasia, and nally to gastric cancer, considered as an in ammation-driven tumor. It has been noted that the beta subunit of H+/K + ATPase was a primary antigen recognized by sera from atrophic gastritis patients [11,12]. In accordance with this, autoantibodies against ATP4B are regarded as serological diagnostic markers for patients with autoimmune atrophic gastritis, which are linked to an increased risk for gastric cancer [13]. Additionally, previous bioinformatics data have suggested that decreased ATP4B expressed in human GC tissue was substantially correlated with poor overall survival in patients with GC [14,15]. Therefore, further investigation into biological function of ATP4B in GC may improve understanding of the molecular mechanisms responsible for providing novel therapeutic targets of GC.
In this study, we intended to reveal potential roles of ATP4B in GC progression. We overexpressed ATP4B in gastric cancer SGC7901 cell line and utilized iTRAQ proteomic technique to identify the proteins modulated by ATP4B. Integrate bioinformatics analyses and Western blot were subjected to validate some of the key effects and proteins regulated by ATP4B in GC cells. This study may provide a scienti c basis for further research on the role of ATP4B in gastric cancer progression and introduce a promising therapeutic target in patients with GC.

Methods And Analysis
Clinical samples and immunohistochemistry (IHC) assay Gastric carcinoma tissue and normal samples were collected from Seventh Medical University of Chinese PLA General Hospital and Beijing Cancer Hospital/Institute. Tissue sections (4 µm thickness) from formalin-xed, para n-embedded specimens were prepared. The protein expression of ATP4B was detected with a mouse monoclonal antibody (Anti-hydrogen/potassium ATPase beta, 2G11, Thermo scienti c, US. 1:300 dilution). A positive reaction was indicated by a reddish-brown precipitate in the nucleus and cytoplasm. Three independent pathologists scored the sections without the knowledge of patients' information.
Cell Culture Human GC cell lines (SGC7901, AGS, BG823) were restored in our laboratory and cultured in Dulbecco's modi ed Eagle's medium (DMEM; Gibco, Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Life Technologies) at 37 °C with 5% CO2. Cell growth was noted at regular intervals each day, with the culture medium changed according to the incubation condition.

Cell Transfection
In order to generate recombinant ATP4B plasmid and condition of ATP4B transfection, PIRES-ATP4B and vector control pIRES (Clontech, US) were constructed in our study. The full length ATP4B cDNA in pIRES was sequenced to con rm the identity and orientation of the ATP4B gene in this construct. GC cells at the logarithmic growth phase were digested with trypsin and respectively inoculated in a six-well plate. Cells were transfected with PIRES-ATP4B or PIRES plasmid when the cell con uence was 80%. In vitro transfection was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instruction.
In order to observe the live GC cells with ATP4B-EGFP overexpressing,based on the empty vector, pEGFP-N1, We designed the primers as follows to form the novel ATP4B-EGFP overexpressing plasmid, F-ATP4B-EGFP: 5'-CCGCTCGAgATGGCGGCTCTGCAGGAGAAG-3' and R-ATP4B-EGFP: 5'-CCCAAGCTTCTTCTCAATCTTGAGTTTGAACTC-3' and the results of some plasmids were identi ed by commercial sequencing (Tianyi Huiyuan Ltd., Beijing, China). By plasmid transfection (pEGFP-N1 and ATP4B-EGFP vectors) and confocol analyses, the live cells with different green uorescence distribution, the cells with pEGFP-N1 transfection, nuclear and cytoplasm green uorescence, while the only cytoplasm green uorescence location was detected in live cells with ATP4B-EGFP transfection. RT-PCR [16] and western blot were also used to examine the expression ATP4B plasmid in GC cells.
Itraq Proteomics SGC7901 GC cells transfected with PIRES-ATP4B or vector for 48 h were harvested and wash twice with PBS. Washed cells were dissolved in RIPA lysis buffer to extract protein. Then, the protein samples were added dithiothreitol to a nal concentration of 10 mM in a water bath for 1 h at 56 °C and then supplemented with iodoacetamide to a nal concentration of 55 mM for an alkylation reaction at room temperature for 1 h in the dark. Every sample was precipitated with four volumes of cold acetone (− 20 °C) were added to the for protein precipitation more than 3 h at − 20 °C. After centrifugation (20000 g, 4 °C, 20 min), the supernatant was discarded and protein precipitation was re-suspended in lysis buffer.
The Bradford method was used to determine the nal protein concentration. 100 µg of protein from each of samples incubated with trypsin and TEAB overnight at 37 °C. Then, the iTRAQ Reagent − 8PLEX Multiplex Kit (Thermo Fisher Scienti c) was used for peptide labeling according to the manufacturer's instructions. Protein samples of SGC7901 with overexpression ATP4B and controls were labeled with 119, 117 iTRAQ tags. The iTRAQ-labeled samples (mixed with buffer) were desalted by Strata X C18 SPE column and dried by vacuum centrifugation. HPLC-MS/MS analysis by using ESI-QUAD-TOF tandem mass spectrometer (Thermo Fisher Scienti c) was applied to identify peptide signal data les. Mascot database (Version 2.3.01, Matrix Science, London, UK) combined with Non-Redundant protein database of NCBI were used to extract proteins abundant pro les and quanti cation information. Differential expressed proteins (DEPs) were identi ed by two-tailed Student's t-tests with Fold Chang > 1.2 and p value < 0.05.

Bioinformatics Analysis
Functional Enrichment Analysis of Gene Ontology (GO) In order to clarify the biological function signi cance correlated with ATP4B-modulated DEPs, we used plug-in ClueGO app [17] in the Cytoscape software (version 3.72, Cytoscape Consortium, New York, NY) to create GO functional categories (biological process, cellular component and molecular function). The GO terms that have a adjust p value < 0.05 were regarded as cut-off criterion and top 15 terms visualized by R software (R3.6.1, https://cran.r-project.org/).

Co-expression Network With Protein-protein Interaction Analysis
To further understand the molecular interactions between the identi ed DEPs, Co-expression network analyzed the protein-protein interactions ((Protein-Protein Interaction Databases. http://www. Genein nity.org/sp/sp_proteininteraction. html). In addition, DEPs were submitted onto STRING online database (http://www.string-db.org/) was to select interacting proteins. Cytoscape software (version 3.72, Cytoscape Consortium, New York, NY) was used to visualize the networks. Meanwhile, hub proteins in PPI network were demonstrated by Cytoscape MCODE and cytoHubba plug-in.

Kyoto Encyclopedia Of Genes And Genomes (kegg) Pathway Analysis
To identify the crucial signaling pathways and diseases related DEPs, KEGG pathway was performed on DEPs via R package clusterPro ler [18] (http://www.bioconductor.org/packages/clusterPro ler/) in R software. We used p value screening threshold of 0.05 to explore enriched pathways. In our study, we chose the top twenty KEGG pathways.

Ingenuity Pathway Analysis
DEPs were analyzed with QIAGEN's Ingenuity Pathway Analysis algorithm (http:www.qiagen.com/Ingenuity, IPA, QIAGEN Redwood City) to identify the hidden biological signi cance of ATP4B in GC cells. Based on the IPA's analysis, interactions network of DEPs associated with cell metabolism, biological function and disease as well as upstream regulatory network were algorithmically generated based on published literature and observed protein expression from IPA experimental data set. Signi cant results were then ranked by -score and p value for prediction the activation status of these processes. |z| > 2 is empirically considered statistical signi cance.

Western Blot Analysis
Western blot analysis Whole cell lysates were prepared from GC cells after 48 h transfecting, and standard western blotting was done according to the protocol from the manufacturer. Protein concentrations were quanti ed via BCA Protein Assay Kit (Thermo Scienti c). 30 µg of extract subjected to polyacrylamide gel electrophoresis.

ATP4B expression level was reduced in paired GC tissues
To overview the expression levels of ATP4B in human gastric tissues, TCGA data from online analytic tools, including The Human Protein ATLAS and GEPIA were used. The data indicated that ATP4B was high-expression in human gastric mucosa (Fig. 1a) and lower expression of ATP4B gene in gastric cancer tissues compared to paired normal tissues was also observed (Fig. 1b). To further validate this result, we analyzed the mRNA expression level of ATP4B in 30 fresh frozen GC samples compared with the paired adjacent normal appearance tissues from Seventh Medical Center of PLA Hospital, and found a decreased expression in GC tissues (Fig. 1c). Moreover, IHC tissue microarray analysis was performed in a cohort including 247 GC cases and 120 adjacent normal tissues from Beijing Cancer Hospital. The results showed that the positive rate of ATP4B expression in tumors was signi cantly less than that of normal tissues (Fig. 1d). From this cohort, only 118 GC patients were enrolled with complete follow-up for Kaplan-Meier analysis, evaluating the possible prognostic value of ATP4B. We found that GC patients with low expression of ATP4B was signi cantly associated with worse survival (Fig. 1e, P < 0.001).

Effects Of Atp4b On Protein Expression In Gc Cells
To investigate the roles of ATP4B in GC progression, we restored ATP4B expression in SGC7901 (adenocarcinoma cell line with lowly ATP4B expression [10]) and detected the alterations of ATP4Bmediated proteins expression in GC cells. Immuno uorescence exhibited the cytoplasmic expression of ATP4B after transfecting with ATP4B-EGFP in SGC7901 (Fig. 2a). RT-PCR and by Western blot validated the stable expression of ATP4B-EGFP in SGC7901 cells (Fig. 2b). ITRAQ proteomics technology is a powerful methodology in the search for disease-speci c biomarkers and gene downstream targets. Therefore, we used iTRAQ proteomics to erect protein expression pro les of over-expression ATP4B in SGC7901 compared with the matched controls (Fig. 2c). All proteins with adjusted p values (Benjamini-Hochberg, FDR) < 0.05 and altered expression levels at a|fold change|> 1.2 were identi ed as DEPs. A total of 293 proteins were differentially expressed in SGC7901 cells with ATP4B overexpression; 145 proteins were upregulated and 148 were downregulated (Fig. 2d).

Atp4b Regulated Mitochondria-related Functional Categories
Gene Ontology Functional Enrichment Analysis was performed on the DEPs in two groups to examine the biological signi cances. Signi cant GO terms were functionally classi ed by biological process (BP), cellular component (CC), and molecular function (MF). In the light of BP category, differential proteins were strongly associated with cell metabolic processes, including energy metabolism, amino acid metabolic processes and cellular lipid catabolism. Other signi cant BP terms were correlated with translation and viral gene expression process (Fig. 3a, Table 1). For the CC category, the differential proteins were predominantly located in mitochondrion including mitochondrial membrane, mitochondrial envelope, mitochondrial matrix, mitochondrial protein complex as well as oxidoreductase complex (Fig. 3b). The prominent MF of differential proteins was involved in oxidoreductase activity and coenzyme binding, enoyl − CoA hydratase activity and fatty-acyl-CoA binding and NAD(P)H/NAD binding, which are associated with energy production and cellular metabolism (especially glucose and lipids metabolism) in the mitochondria (Fig. 3c). Collectively, these data suggest that ATP4B acts a prominent role in regulating the mitochondria bio-function and energy metabolism. To further predict which regulatory roles and biological functions/diseases might be activated or inactivated by the altered protein expression pro ling after ATP4B overexpressing in GC cells, IPA upstream regulatory effect analysis and downstream function analysis were conducted based on IPA literature. Upstream regulatory effect analysis identi ed the potential upstream regulatory network and downstream function in the DEPs network. PML, STAT4 and other molecules were predicted as upstream regulators. Top regulatory effect network identi ed FGF7, MAPK9, PML as upstream regulators that control the expression of BAX, CDKN2A, EGFR, FASN, HSPB1, NDRG1, S100A10, SCD, SQSTM1. Finally, related downstream anticancer effects functions were activated (Fig. 4a). Consistently, the results of disease/function analysis also indicated that ATP4B-induced protein expression alterations were remarkably associated with regulating cancer-related biological behaviors: repressing cell invasiveness and tumor metastasis, promoting cell death and cell apoptosis (Fig. 4b, Table 1). In line with the data of GO signi cant terms, the changed functions concluding metabolism of glycolipid and energy production were also observed after ATP4B overexpression in GC cells. As shown in Fig. 4b, we presented the regulation interaction between correlative DEPs and invasion of cells after ATP4B overexpression in GC cells.

Construction Of Molecular Networks
We further conducted biological protein-protein interaction network to predict the pivotal proteins regulated by ATP4B. According to the data of GO and IPA analysis, co-expression network related to cell apoptosis and metabolism were constructed. In total, the co-expression network contained 41 nodes. P53 and RELA were regarded as the central genes, exhibiting an important interaction between p53-related signaling and NF-κB pathway (Fig. 5a). Following the construction of the PPI network, one hub-network included 119 nodes and 515 edges and the top ten core proteins (EGFR, UBC, PGK1, ALB, IDH1, NPM1, STAT3, EEF1G, CDKN2A, ALDH18A1) were presented (Fig. S1a). Additionally, we analyzed the interrelationships among the experimental proteins and the IPA database proteins via molecular network analysis. Intriguingly, the top-ranked network diagram indicated a major role of the DEPs in the regulation of mitochondrial metabolism (Fig.S1b).
ATP4B-modulated downstream effectors were signi cantly correlated with p53/ NF-κB/ mitochondria signaling pathway KEGG pathway enrichment analysis was performed to identify the potential mechanism of ATP4Bregulated bio-function associated with GC progression in our study. It revealed that ATP4B-modulated DEPs were primarily involved in pathways associated with cell metabolic pathways including: Biosynthesis and metabolism of amino acids, Carbon metabolism, Sulfur metabolism, Fatty acid metabolism and degradation, Citrate cycle (TCA cycle). In addition, p53 signaling pathway and PPAR signaling pathway (mainly regulated lipid metabolism) are also important pathways capable of regulating cancer initiation and development (Fig. 5b).
To our knowledge, the p53 pathway and NF-κB are mutually repressed, involving in suppressing the tumorigenesis and development of cancer [19]. Besides, p53 gene and NF-κB gene have been reported to regulate tumor cell metabolism, mitochondrial function [20,21]. In order to prove the results of our bioinformatics analysis above, we utilized western blot analysis to validate selected signi cant DEPs. The results showed that the protein levels of p53, p21 and p16 increased following ATP4B overexpression SGC7901 cells and the apoptosis related genes, such as Bax, Bid were increased while anti-apoptotic proteins Bcl-2 decreased (Fig. 5c). Moreover, our analysis also showed that the protein level of p65 and p-ΙκΒα, pSTAT3, CD44 were decreased after upon ATP4B overexpressed in SGC7901 indicating ATP4B inhibited NF-κΒ pathway (Fig. 5c). In agreement with this, we found the same corresponding protein expression after overexpressing ATP4B in AGS and BGC823 (Fig.S2). Theoretically, we believed that restoration of ATP4B expression in GC cells regulated mitochondrial function and cell death through activation of p53 pathway along with the inhibition of NF-κΒ/CD44 pathway, determining the inhibitory role of ATP4B in progression of GC (Fig. 5d).

Discussion
The initiation and development of GC is a multi-stage, heterogeneous and multifactorial pathology process involving numerous genetic, epigenetic and environmental factors alterations [22]. Recently, studies have reported that the ATP4B gene is downregulated in GC, which plays a negative role in the progression of GC [6,15]. Restoring ATP4B expression in GC cells may heighten the inhibitory effects of chemotherapeutic drugs on GC cell growth [10]. Consistent with these studies, our current study veri ed the lower ATP4B expression in GC tissues than normal gastric mucosal via TCGA data, presenting a worse survival. ITRAQ proteomics and bioinformatics analysis were used to further investigate target pro les and the signaling pathway related to ATP4B regulation, unveiling the potential molecular roles of ATP4B in gastric cancer progression.
GO analyses in our study revealed a large proportion of ATP4B-DEPs were closely correlative to mitochondria-related GO terms and involved in energy production and cellular metabolism by affecting the mitochondrial enzymes. Mitochondria are complex organelles whose major functions are energy conversion and production ATP through OXPHOS system [23]. It is well-known that mitochondrial dysfunctions have broad impacts on suppression of tumor growth as they could cause the aberrant bioenergy metabolism of cancer cells [24,25]. The most extensively studied metabolic alterations in cancer are glucose metabolic reprogramming. Besides altered glucose metabolic phenotype, biosynthesis and utilization of lipids abnormality as well as amino acid metabolism changes are also recognized as the one of the most common metabolic features of cancer cells. In the last few decades, increasing studies suggested that dysregulation of lipid metabolism, especially for the reactivation of de novo fatty acid products, were essential for tumor progression [26,27]. High rate of glucose uptake and deregulated lipid metabolism have been recognized as a pivotal hallmark of cancer [28]. GC cells and normal cells exhibit metabolic differences in glucose metabolism as well as the metabolism of lipids and amino acid [29]. In the present study, the GO molecular function showed that the dominant biological roles of ATP4Bmodulated DEPs regulated the enzymes activity of lipids and glucose metabolism, such as acyl-CoA hydrolase, NADH dehydrogenase (ubiquinone) activity, oxidoreductase activity. In agreement with the role of ATP4B in regulating cellular energy metabolism, GO cellular component categories for DEPs were enriched in mitochondrial protein complex and mitochondrial envelope and inner membrane, and it is the organelles where fatty acid β-oxidation, TCA cycle occur. Comprehensively, high ATP4B expression leads to mitochondrial function and energy metabolism alterations in gastric cancer.
Based on IPA analysis, our study theoretically showed that ATP4B gene exhibits an inhibitory role in GC cells, repressing cell invasion, metastasis and inducing apoptosis, preventing the advancement of gastric cancer. In addition, ATP4B-mediated functions were also associated with regulating cell metabolism, such as cell energy production and metabolism of glycolipid. The results of hub protein-protein interaction and IPA network analysis also indicated that a few highlighted hub proteins mainly took part in the energy metabolism process. Co-expression network analysis showed an important network related to p53 and NF-κΒ pathway involved in regulating cancer progression. KEGG pathway analysis revealed that the top 20 pathways were relevant to cell metabolism pathways and p53 signaling pathway.
P53 gene, acted as a tumor suppressor, mainly controls cell cycle progression and cell death which play key roles in tumor suppression [30]. There are converging numbers of studies showing that p53 pathway also regulates cell metabolism-associated phenotype modi cations, not only the control of glycolysis and mitochondrial respiration, but the effect on lipid and nucleotide metabolism [31]. P21, also known as cyclin-dependent kinase inhibitor 2A, is a well-known p53 target gene and its protein products link the p53 and cell cycle arrest [19]. Loss of p21 may predict poor outcome in gastric carcinoma which increases histologic grade or depth of invasion and lymph node and peritoneal metastases [32]. P16 is a cell cycleassociated protein, it has been reported that the activation of p16 led to potent antitumor e cacy in gastric cancer [33]. It has been universally acknowledged that the intrinsic pathway which may involve p53. P53 has a dual action of promoting anti-apoptotic members activities as well as down-regulation anti-apoptotic Bcl-2 family members, triggering mitochondria-mediated apoptosis pathway [34,35].
Western blot veri cation in our study illustrated that ATP4B expression activated p53 pathway and promoted cell apoptosis.
Previous studies have veri ed that p53 and NF-κΒ mutually repressed and overexpression of wildtype p53 inhibited NF-κΒ activity, therefore induced apoptosis. Our co-expression network analysis presented a key interaction between p53-related signaling and NF-κB pathway. Nuclear factor kB (NF-kB), a nuclear transcription factor, is composed of ve family members: RelA (p65), p105/p50 (NF-κΒ1), p100/p52 (NF-κΒ2), c-Rel, and RelB. NF-κΒ is essential in various biological processes including cell survival, proliferation, apoptosis, adhesion, angiogenesis and in ammation [36]. Activation of NF-κB mainly occurs via phosphorylation of IκB proteins including ΙκΒα [37]. It has reported that NF-κB is constitutively activated in gastric cancer tissues, inducing angiogenesis, cell proliferation, metastasis and evasion of apoptosis [38,39]. CD44 is a target gene of NF-κΒ pathway; overexpression CD44 in cancer cells can reroute NF-κB pathway leading to cancer progression and malignancy [40,41]. STAT3 serves a role as a signal transducing molecule between CD44 and NF-κB. pSTAT3 an active form of STAT3 was positively associated with a poor prognosis for patients with gastric cancer [42]. In accordance with these ndings, our study exhibited the reduced expression of NF-ĸB, p65 and pΙκΒα, CD44, pSTAT3 upon restoration of ATP4B, suggesting that ATP4B regulating the NF-κΒ/CD44 pathway.

Limitations
There are some limitations in our current study. Although we have identi ed several ATP4B-mediated functions and pathways in gastric cancer cells by using bioinformatics analysis, there are no experiments to verify the relationship between ATP4B gene and these terms. Future experimental in vitro and in vivo studies are needed to better substantiate and validate the speci c roles and regulatory mechanism of ATP4B in gastric cancer progression. Furthermore, the relevant detail by which ATP4B modulates the mitochondrial metabolic pathway remains unknown and should be addressed in future studies.

Conclusion
In summary, our results validate the lower ATP4B expression in GC tissues than normal gastric mucosal; patients with GC exhibits a worse overall survival. The comprehensive ATP4B proteomic changes in gastric cancer and its biological functions are closely with energy production and cell metabolism. Furthermore, ATP4B plays an anticancer effect in gastric cancer cells most likely by regulating p53/ NF-κΒ/mitochondrial pathway. This study provides the useful resources for further understanding the potential mechanisms of ATP4B responsible for repressing the progression in gastric cancer.