| BackgroundGastric cancer is one of the most common cancers worldwide,ranking fifth in global cancer incidence and fourth in mortality.Due to the lack of effective early diagnosis methods,gastric cancer patients are usually found at an advanced stage,which greatly reduces the fiveyear survival rate of gastric cancer patients.Helicobacter pylori infection is one of the recognized causes of gastric cancer and plays an important role in the development of chronic superficial gastritis-atrophic gastritis with intestinal metaplasia-dysplasia-gastric cancer,but the specific mechanism remains to be further studied.This paper mainly studies the role and mechanism of ferroptosis mediated by the activation of Wnt/β-catenin signaling pathway by Helicobacter pylori in gastric malignant transformation.The Wnt/β-catenin signaling pathway is a highly conserved signaling pathway in the evolution of species,and plays a crucial role in the early development of animal embryos,organ formation,tissue regeneration and other physiological processes.In addition,the Wnt/β-catenin signaling pathway is also involved in the pathological process of tumor occurrence and development,including tumor cell proliferation and apoptosis,invasion and migration,self-renewal of tumor stem cells,and chemotherapy resistance of tumors.Ferroptosis is a novel form of programmed cell death discovered in recent years,characterized by iron-dependent accumulation of lipid peroxides.In the process of ferroptosis,the antioxidant capacity of cells decreases,the redox level is unbalanced,and then the lipid peroxidation reaction occurs in the cell membrane,which damages the integrity of the cell membrane and leads to cell death.Ferroptosis is closely related to the occurrence and development of various tumors,and many studies have confirmed the important role of ferroptosis in killing cancer cells and inhibiting the growth of cancer cells.Cytarabine,cisplatin,doxorubicin and other chemotherapeutic drugs combined with ferroptosis inducers have a significant synergistic effect on their antitumor activity.Therefore,in-depth study of the molecular mechanism of the ferroptosis pathway is of great significance for the treatment of tumors and the development of anticancer drugs.At present,there is no research on the relationship between Wnt/β-catenin signaling pathway and ferroptosis.Therefore,we investigated whether Wnt/β-catenin signaling pathway regulates ferroptosis and whether ferroptosis regulated by Wnt/β-catenin signaling pathway plays a role in the occurrence and development of gastric cancer.The role of Wnt/β-catenin signaling pathway in regulating ferroptosis was studied in detail.ObjectivesTo explore the relationship between Wnt/β-catenin signaling pathway and ferroptosis in the process of Helicobacter pylori infection,and to clarify the mechanism of Helicobacter pylori activation of Wnt/β-catenin signaling pathway to regulate ferroptosis in gastric malignant transformation.Enhancing the chemosensitivity of tumors provides a new strategy.Methods and Results(1)Inhibition of Wnt/β-catenin signaling pathway can specifically enhance the sensitivity of gastric cancer cells to ferroptosisCCK-8 cell viability assay and flow cytometry cell death assay showed that the use of Wnt/β-catenin signaling pathway inhibitor LF3 could specifically enhance the sensitivity of gastric cancer cells to ferroptosis.MDA level detection,ELISA experiment and lipid reactive oxygen species level detection showed that inhibiting the Wnt/β-catenin signaling pathway could increase the level of cellular lipid peroxidation,thereby inhibiting the resistance of gastric cancer cells to ferroptosis.(2)TCF4,a key transcription factor in the Wnt/β-catenin signaling pathway,inhibits ferroptosis in gastric cancer cellsAnalysis of TCF4 expression in TCGA database,GEO database and clinical specimens showed that TCF4 expression was elevated in gastric cancer,and the high expression group had a poor prognosis.Then,by constructing TCF4 gene knockout cell lines and TCF4 overexpressing cell lines,and detecting the ferroptosis sensitivity and lipid peroxidation level of control cells,TCF4-KO cells and TCF4 overexpressing cells,it was found that TCF4 knockout can increase gastric cancer.The level of lipid peroxidation in gastric cancer cells increases the sensitivity of gastric cancer cells to ferroptosis;while overexpression of TCF4 can reduce the level of lipid peroxidation in gastric cancer cells,resulting in gastric cancer cells’ resistance to ferroptosis.(3)TCF4 inhibits ferroptosis in gastric cancer cells by regulating GPX4Through ChIP Seq sequencing,it was found that TCF4 can bind to the promoter region of the core gene GPX4 of ferroptosis.The expression of GPX4 was detected in the TCGA database,GEO database and clinical specimens,and it was found that the expression of GPX4 was increased in gastric cancer.Clonal formation,EdU,scratch assay,and transwell assay confirmed that knockdown of GPX4 could inhibit the proliferation,invasion,and migration of gastric cancer cells.By constructing GPX4 gene knockout gastric cancer cell lines,and overexpressing TCF4 in control cells and GPX4-KO cells,respectively,the level of MDA and the colony formation ability of gastric cancer cells were detected,and it was found that overexpression of TCF4 could reduce the level of lipid peroxidation in gastric cancer cells,enhanced the ability of gastric cancer cell colony formation;when GPX4 was knocked out,the effect of overexpression of TCF4 on gastric cancer cell ferroptosis was abolished.By detecting the sensitivity of gastric cancer cells treated with LF3 or knocking out TCF4 to Erastin and RSL3,respectively,it was found that gastric cancer cells had different sensitivities to ferroptosis induced by Erastin and RSL3,confirming that TCF4 regulates ferroptosis in gastric cancer cells in a GPX4-dependent manner.(4)β-catenin/TCF4 binds to the promoter region of GPX4 in the form of a transcription complex to regulate its transcriptionReal-time quantitative PCR and Western Blot experiments confirmed that after overexpression of TCF4,the mRNA and protein levels of GPX4 were significantly increased;after TCF4 was knocked out,the mRNA and protein levels of GPX4 were significantly decreased.Dual-luciferase assay and chromatin immunoprecipitation assay confirmed that TCF4 can directly bind to the promoter region of GPX4 and regulate its transcription.Then,in order to explore whether β-catenin is involved in the regulation of GPX4 by TCF4,realtime quantitative PCR and Western Blot experiments were performed after knockdown of βcatenin,and it was found that the mRNA and protein levels of GPX4 were significantly reduced.After using inhibitors or transfecting TCF4 mutant plasmids,the binding between βcatenin and TCF4 was abolished,and then real-time quantitative PCR and Western Blot experiments were performed.(5)Inhibition of TCF4 in vitro can enhance the sensitivity of gastric cancer cells to cisplatinThe detection of CCK-8 cell activity showed that knockout of TCF4 enhanced the sensitivity of gastric cancer cells to cisplatin,while overexpression of TCF4 reduced the sensitivity of gastric cancer cells to cisplatin.MDA level detection,ELISA experiments found that cisplatin can increase the level of cellular lipid peroxidation.(6)Inhibition of Wnt signaling pathway in vivo can enhance tumor sensitivity to cisplatinSubcutaneous tumorigenesis experiments in nude mice confirmed that knocking out TCF4 in vivo or using the Wnt/β-catenin signaling pathway inhibitor LF3 to inhibit the Wnt/β-catenin signaling pathway could increase the level of tumor lipid peroxidation and enhance tumor response to cisplatin.Platinum sensitivity promotes cisplatin-induced ferroptosis.(7)Helicobacter pylori infection can up-regulate the expression of GPX4 through TCF4Helicobacter pylori infection of gastric cancer cells detected intracellular MDA,lipid reactive oxygen species,reduced glutathione/oxidized glutathione,and the results showed that Helicobacter pylori infection could inhibit the level of lipid peroxidation in tumor cells and increase The ratio of reduced glutathione to oxidized glutathione in tumor cells affects the occurrence of ferroptosis in tumor cells.Real-time quantitative PCR and Western Blot experiments confirmed that Helicobacter pylori infection could promote the mRNA and protein expressions of TCF4 and GPX4 in a time-and concentration-dependent manner,and TCF4 mediated the up-regulation of GPX4 expression by Helicobacter pylori.The immunohistochemical staining of Helicobacter pylori mice by gavage showed that the expression of TCF4 and GPX4 increased in the Helicobacter pylori gavage group.The realtime quantitative PCR detection of Helicobacter pylori positive and negative human gastritis samples showed that the expression of TCF4 and GPX4 in the positive group was increased.ConclusionThis study found that Helicobacter pylori can activate the Wnt/β-catenin signaling pathway,upregulate the expression of the transcription factor TCF4,and then promote the transcription of GPX4 through the β-catenin/TCF4 transcription complex,thereby inhibiting the level of lipid peroxidation in gastric cancer cells.Inhibits the occurrence of chemotherapymediated ferroptosis.Targeting the Wnt/β-catenin signaling pathway by using inhibitors or knocking out TCF4 can inhibit the expression of the core ferroptosis gene GPX4 and promote the sensitivity of gastric cancer cells to chemotherapy-induced ferroptosis.Our study provides a new mechanism for transcriptional regulation of ferroptosis and a new way to enhance the efficacy of chemotherapy drugs such as cisplatin. |
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