Preliminary Study On The Regulation Of Ferroptosis By CST1 Through GPX4 To Promote Gastric Cancer Progression

Objective:Elucidation of the role of cysteine protease inhibitor SN(CST1)in promoting gastric cancer progression through the regulation of ferroptosis by glutathione peroxidase 4(GPX4),a membrane lipid repair enzyme,and its mechanism.Methods:(1)Firstly,185 paired pairs of gastric cancer and paraneoplastic tissues were subjected to immunohistochemical detection of CST1 protein expression levels,immunohistochemical staining maps of CST1 were analysed and immunohistochemical scores were statistically analysed.(2)Primary gastric cancer tissues and corresponding non-cancerous paraneoplastic tissues from 9 5 gastric cancer patients who underwent gastrectomy without neoadjuvant therapy were collected,and after completion of follow-up and clinicopathological and prognostic analyses,the correlation between CST1 protein and clinicopathological factors,prognosis and potential clinical applications of gastric cancer patients were investigated.(3)To analyse the correlation between CST1 protein expression levels and gastric cancer using the GEO database in the datasets GSE54129,GSE66229,GSE79973,GSE26899,GSE13911 and GSE19826.(4)The relationship between high and low CST1 protein expression and prognosis of gastric cancer patients was analysed using the Kaplan-Meier Plotter online database.(5)The association between gastric cancer patients with high CST1 protein expression and prognosis was analysed by univariate and multifactorial Cox regression models to screen for independent predictors of gastric cancer prognosis.(6)Immunoprecipitation was used to enable CST1-specific antibodies to interact with proteins from MKN4 5 gastric cancer cells,which were then analysed by liquid chromatography-mass spectrometry and finally silver staining to identify proteins that might interact with CST1.(7)KEGG pathway enrichment analysis was performed on the enriched proteins to explore the signalling pathways that CST1 may affect.(8)To demonstrate that protein-protein interactions between CST1 and GPX4 may induce malignant progression of gastric cancer.(9)To investigate the expression levels of GPX4 protein in gastric cancer tissues using protein immunoblot analy0is assays using five paired gastric cancer tissue and adjacent normal gastric mucosal tissue samples.(10)Ninetyfive pairs of gastric cancer and paracancer tissues were randomly selected from 1 8 5 paired pairs and immunohistochemical staining was used to assess the expression levels of CST1 and GPX4 in primary foci of gastric cancer patients.(1 1)HEK293T cells transfected with flag-tagged CST1 expression plasmid and myc-tagged GPX4 ex pression plasmid were treated with cycloheximide and MKN4 5 cells transfected with CST1-interfering lentivirus were treated with 26S proteasome inhibitor MG132,respectively,to investigate whether CST1 has an effect on the stability of GPX4 protein.(12)To investigate whether CST1 affects the ubiquitination of GPX4 using immunoprecipitation and immunoblot analysis.Results:(1)CST1 protein is highly expressed in gastric cancer tissues,and the lower the degree of tumour differentiation,the higher the expression.(2)CST1 expression level correlated with clinicopathological factors such as the degree of tumour differentiation and lymph node infiltration.(3)In the GEO source dataset,CST1 was significantly highly expressed in gastric cancer tissues.(4)In the KM Plotter database,the expression level of CST1 protein was negatively correlated with prognosis.(5)The results of univariate and multifactorial Cox regression model analysis showed that high expression of CST1 protein was an independent risk factor affecting the prognosis of gastric cancer patients.(6)CST1 can interact with 63 proteins,most likely affecting the ferroptosis signalling pathway.(7)Interaction of CST1 with GPX4 induces malignant progression of gastric cancer.(8)GPX4 protein expression was elevated in gastric cancer tissues and gastric cancer cells and positively correlated with CST1 expression levels.(9)CST1 protein enhances the protein stability of GPX4 by inhibiting its ubiquitination.Conclusions:CST1 expression is elevated in gastric cancer and may promote the progression of gastric cancer by binding to GPX4 and regulating ferroptosis.