Investigating Biological Function And Mechanism Of HAP1 In The Carcinogenesis Of Gastric Cancer

BackgroundGastric cancer is the most familiar malignant tumor of digestive tract worldwide,with insidious onset,low early detection rate,and poor prognosis,which poses a serious threat to human life and health and is the strategic focus of current cancer prevention and treatment research.Our country is a high incidence and high death area of stomach cancer,according to the 2022 National Cancer Report released by the National Cancer Center,gastric cancer ranks the third in both morbidity and mortality.Gastric cancer is a common multifactorial and complex malignancy,its pathogenesis involves many factors such as environment and heredity.So far,there has been no breakthrough in the studies on the pathogenesis and early diagnosis of gastric cancer,which cannot meet the needs of effective clinical control of gastric cancer.Therefore,it is particularly urgent to reveal the pathogenesis and development of gastric cancer and seek new targets for diagnosis and treatment,which has important public health significance to reduce the global burden of gastric cancer.Huntingtin-associated protein 1(HAP1)is the first protein that interacts with Huntington’s protein,which is involved in gene transcription regulation,vesicle transport,membrane endocytosis,signal transduction,and inclusion body formation.Studies have shown that HAP1 is closely related to Huntington’s disease,and Huntington’s disease is negatively correlated with the occurrence of tumors.In recent years,scientists have paid more and more attention to the relationship between HAP1 and cancer,mainly involving breast cancer,pancreatic cancer,acute lymphoblastic leukemia,and so on.Studies have shown that overexpression of HAP1 can affect the migration,invasion,and apoptosis of breast cancer cells,suggesting that HAP1 may be a new tumor suppressor gene for breast cancer.HAP1 is associated with the occurrence and development of a variety of tumors,but its tissue specificity and biological function are not clear,especially in digestive system tumors.HAP1 is mainly expressed in the nervous system,and also in the internal digestive system,while it is differentially expressed in different organs and mucous membranes of the gastrointestinal tract.The expression of HAP1 in the stomach is mainly concentrated in the pylorus,especially in the base of the gland of the pylorus gland.The different specific expression of HAP1 in the stomach suggests that HAP1 is related to the biological characters and functions of the stomach,but its molecular mechanism is still unclear.At present,the relationship between HAP1 and tumorigenesis and development has received increasing attention,but there are few studies on gastric cancer.In this study,bioinformatics means were used to analyze the difference of HAP1 expression in the gastric cancer database and the impact of HAP 1 on the overall survival rate of gastric cancer patients.The results showed that the expression of HAP1 was down-regulated in gastric cancer,and the overall survival rate of patients with low expression of HAP 1 was lower than that of patients with high expression of HAP1.Based on the conclusion of bioinformatics analysis,immunohistochemistry,Western Blot,and Real-time quantitative PCR were used to detect the expression of HAP1 in gastric cancer tissue samples and gastric cancer cell lines.The stable transfected HAP1 gastric cancer cell lines were constucted to explore the effects of HAP 1 on the proliferation,migration,and apoptosis of gastric cancer cells in vitro.Meanwhile,a nude mouse subcutaneous tumorigenic animal model was established to verify the effect of HAP1 on gastric cancer at the experimental animal level.To study the molecular regulatory mechanism of HAP 1 on the above biological functions,Western Blot was used to detect the expression levels of key proteins in the signaling pathways related to cell proliferation,cell cycle,cell migration and invasion,and cell apoptosis.HAP1 may directly or indirectly regulate the corresponding signaling pathways through these key proteins.It affects the occurrence and development of gastric cancer.This study preliminarily explored the key biological processes and mechanisms that HAP1 may be involved in the occurrence and development of gastric cancer and provided the theoretical basis for finding new targets and therapeutic means for the clinical diagnosis and treatment of gastric cancer.Methods1.Bioinformatics method was used to analyze the difference of HAP 1 expression in the gastric cancer sample data set of the UCSC Xena database and TNMplot database;The effect of HAP1 on the overall survival rate of gastric cancer patients was analyzed through the Kaplan-Meier Plotter database.2.Immunohistochemical staining,Western Blot,and real-time fluorescence quantitative PCR were used to detect the difference in the expression of HAP1 in normal gastric mucosa and gastric tumor tissues of patients with clinical gastric cancer,and the difference in the expression of HAP1 in gastric mucosal epithelial cells GES-1 and various gastric cancer cells cultured in vitro.3.The effect of overexpression of HAP 1 on the protein expression profile of gastric cancer cells was detected by iTRAQ proteomics.4.The effect of overexpression of HAP1 on the proliferation of gastric cancer cells was detected by cell counting,EdU flow cytometry,and colony formation assay;cell cycle synchronization and flow cytometry were used to detect the effect of overexpression of HAP 1 on the cell cycle of gastric cancer cells;The effect of overexpression of HAP1 on tumorigenesis in nude mice was detected by subcutaneous tumorigenesis test in nude mice;Western blot was used to detect the expression level of key proteins in cell proliferation and cell cycle-related signal pathways.5.The effect of overexpression of HAP1 on the migration and invasion of gastric cancer cells was detected by the wound healing assay and Transwell test;Western blot was used to detect the expression level of key proteins in signal pathways related to cell migration and invasion.6.Gastric cancer cells were treated with chemotherapy drugs or glucose starvation,and the effect of overexpression of HAP1 on apoptosis of gastric cancer cells was detected by annexin V-FITC/PI staining and flow cytometry;Western blot was used to detect the expression level of key proteins in apoptosis-related signal pathways.Results1.The expression of HAP1 was down-regulated in clinical gastric cancer tissues in the database.The overall survival rate of patients with low expression of HAP1 was lower than that of patients with high expression of HAP1.2.The expression of HAP1 was down-regulated in clinical gastric cancer tissues and gastric cancer cells cultured in vitro.3.iTRAQ proteomics technology screened 30 proteins with significant differences.HAP1 may regulate the Ca2+signaling pathway through InsP3R1 and ITPRIP.4.Overexpression of HAP 1 inhibits the proliferation of gastric cancer cells and leads to cell cycle arrest,and inhibits the ability of subcutaneous tumorigenesis in nude mice;There was no significant difference in the protein level of AKT in gastric cancer cells overexpressing HAP1,but the protein level of p-AKT(Ser473)and cyclin E2 and cyclin A2 decreased.5.Overexpression of HAP1 inhibits the migration and invasion of gastric cancer cells;In gastric cancer cells overexpressing HAP1,the protein level of E-cadherin was up-regulated,while the protein level of Vimentin and Snail1 were down-regulated.6.Overexpression of HAP1 promotes the apoptosis of gastric cancer cells under chemotherapy drug treatment or glucose starvation;The level of Bcl-2 protein in gastric cancer cells overexpressed-with HAP1 is down-regulated,but the level of Bax and Bcl-xl protein is not significantly different,and the level of Caspase-3 and Caspase-12 protein is up-regulated.ConclusionsThe expression level of HAP 1 was down-regulated in clinical gastric cancer tissues and gastric cancer cells;HAP1 inhibits the proliferation and cell cycle of gastric cancer cells by inhibiting AKT signal pathway and cell cycle-related proteins;HAP1 inhibits cell migration and invasion of gastric cancer cells by inhibiting the transcription factor Snail1;HAP1 promotes the apoptosis of gastric cancer cells by regulating the Bcl-2 family.To sum up,HAP1 may be a potential tumor suppressor gene for gastric cancer.This study provides a theoretical basis for finding new targets for clinical diagnosis and treatment of gastric cancer.