Abnormal Expression Of ATP4B Affects Gastric Mucosa Cell Energy Metabolism And Gastric Carcinogenesis Via Regulating P53/SCO2 Signaling Pathway

Background and objectivesGastric cancer remains one of the most common gastrointestinal malignancies with high morbidity and mortality worldwide.H+-K+-ATPase Transporting Subunit Beta(ATP4B)is mainly expressed in the parietal cells of the stomach and serves as a gastric function gene.It has been reported that decreased ATP4B expressed in human GC tissue was substantially correlated with poor overall survival rate in patients with GC via bioinformatics analysis in Cancer Genomics database.Our previous study showed that restoration of ATP4B expression played an anti-cancer effect without systematic mechanical investigation of ATP4B gene on gastric cancer occurrence and development.This study was aimed to further investigation into the underlying biological functions and mechanisms of ATP4B in Gastric carcinogenesis and by interfering or restoring ATP4B expression.Methods1.The differential expression between gastric cancer tissue and gastric mucosal tissue ATP4B was explored through Gene Expression Profiling Interactive Analysis and The Human Protein ATLAS online databases.2.Immunohistochemistry was used to test the change of ATP4B expression in gastric cancer tissue samples.Then,the correlation between the expression of ATP4B expression and clinicopathologic features along with prognosis in GC patients was evaluated.3.Western blot was used to verified the expression of ATP4B in immortalized gastric mucosal epithelial cell line and common gastric cancer cell lines.CCK-8 tests,scratches tests,Transwell tests and TUNEL tests were performed in MGC803 cell line to detect the effect of ATP4B expression on proliferation,migration and invasion,and apoptosis level of gastric cancer cell.At the same time,siRNAs that interfere with ATP4B were constructed,and siATP4B transfection experiments were carried out in GES-1 and AGS cell lines.After verifying interference efficiency by Western blot,the above-mentioned cell function tests were used to exploration the changes of biological behavior after interference ATP4B expression in AGS and GES-1 cell lines.4.Using Cytoscape and R bioinformatics software to analyze the underlying biological functions and processes of ATP4B-regulated downstream different proteins in GC tumorigenesis and development via functional and the path cluster.Then,it was verified by Western Blot,RT-qPCR assays,mitochondrial function tests,and cell phenotype tests.Results1.The expression level of ATP4B was decreased in gastric tissues and cell lines.Loss of ATP4B expression was associated with lower differentiation,lymph node metastasis,TNM stages.Overall,ATP4B expression is positively correlated with overall survival time in patients with gastric cancer.2.Restoration of ATP4B expression in gastric cancer cells induced apoptosis and suppressed cell proliferation,migration and invasion,whereas ATP4B silencing promoted the malignant phenotype of gastric cancer.3.Bioinformatics analysis showed that ATP4B-mediated downstream proteins were primarily involved in mitochondrial functions.KEGG pathway analysis showed that differential proteins were also significantly enriched in the p53 signaling pathway except the enrichment of energy metabolism.Cell function assays suggest that ATP4B can be used as a cancer-suppressing gene to participate in gastric mucosal cancer,restore the expression of ATP4B in gastric cancer cells can affect the proliferation and apoptosis activity of gastric cancer cells by activating the P53 pathway.The expression of ATP4B in gastric cancer reduced intracellular ATP and lactic acid production and increased ROS generation;overexpressing ATP4B and siP53 co-transfection in MGC803 increased ATP and lactic acid generation and decreased intracellular ROS compared with the ATP4B overexpression in gastric cancer cell.Furthermore,the results of RT-PCR and Western blot assays suggested that restoration the expression of ATP4B in gastric cancer cells can activated the p53/SCO2 pathway which was involved in regulating cell energy metabolism and cancer.ConclusionOur results verify the lower ATP4B expression in gastric tissues and cell lines;patients with GC exhibits a worse overall survival.Abnormal expression of ATP4B affects gastric mucosa cell energy metabolism and gastric carcinogenesis via regulating p53/SCO2 pathway.This study provides the useful resources for further understanding the potential mechanisms of ATP4B responsible for repressing the progression in gastric cancer and suggested that ATP4B maybe used as potential prognostic markers in patients with gastric cancer.