| Objectives: Iron ions are involved in many metabolic pathways in cells,so the homeostasis of iron ions is vital to the normal life of cells.The purpose of this study was to explore the relationship between iron and gastric cancer and its key mechanism by verifying the effect of iron overload on the proliferation,migration and invasion of gastric cancer cells and analyzing the changes of iron metabolism mode in patients with gastric cancer metastasis.Meanwhile,we constructed a prognostic model of iron overload in gastric cancer patients,in order to provide beneficial guiding significance for the prognosis and treatment of gastric cancer patients.Methods: MTT assay was used to detect the effect of iron overload on the proliferation of HGC-27,BGC-823 and MGC-803 cells.At the cell level,wound healing and transwell assay were used to detect the effect of iron overload on the migration and invasion of three cell lines.At the level of transcription and translation,the effects of iron overload on the epithelial-mesenchymal transition related biomarkers of the three cells were detected by real-time PCR and western blot.RNA-seq identifies potential signaling pathways altered after iron overload,and verified by western blot.Pan cancer expression of iron uptake related genes TFRC and DMT1 analyzed by bioinformaticsmethods.The expression of TFRC and DMT1 proteins in gastric cancer tissues was also determined by bioinformatics methods.The Consensus Clustering was used to construct molecular subtypes of iron metabolism related gastric cancer,and R was used to perform survival analysis,clinicopathological features analysis,molecular biological features analysis,and tumor microenvironment features among the different molecular subtypes.Principal component analysis to construct an iron metabolism signature gene score for each gastric cancer patient,R was used to analyze the survival differences of patients in high and low scoring groups and the relationship with the molecular subtypes of iron metabolism.Finally,differences in PD-L1 expression,tumor mutational burden,and microsatellite instability between high and low scoring groups were analyzed.A gastric cancer iron overload prognostic model was constructed using the lasso regression method,and the risk score was calculated for each patient with gastric cancer.After that,gastric cancer patients were divided into high and low risk groups using the median value of the risk score as the cutoff value,and this model was validated in the test set.Survival analysis,clinicopathological features analysis,molecular biological features analysis,and tumor microenvironment features were performed on the population in the high and low risk groups using R.The accuracy of the model was verified by a time-dependent receiver operator characteristic curve.The accuracy of nomograph is verified by decision curve analysis.Results:1.Iron overload promoted the proliferation ability of human gastric cancer cells HGC-27,BGC-823,MGC-803,in which the proliferation ability of HGC-27 reached the maximum at an iron ion concentration of 600 ?M and that of BGC-823,MGC-803 at an iron ion concentration of 800 ?M.Meanwhile,iron overload promoted the migration ability of human gastric cancer cells HGC-27,BGC-823 as well as MGC-803,the migration ability of the three cell lines reached the maximum at the iron ion concentration of 400 ?M.The invasion ability of the cells increased significantly at the ferric ion concentration of 600 ?M.Iron overload upregulated Vimentin,Twist1,Snail1,N-cadherin genes and downregulated E-cadherin genes in human gastric cancer cells HGC-27,BGC-823,MGC-803.Meanwhile,iron overload promoted vimentin,fibronectin,Twist1,Snail1,N-cadherin protein expression and inhibited E-cadherin protein expression in human gastric cancer cells HGC-27,BGC-823 as well as MGC-803.Pathway enrichment of the differential genes obtained from RNA-seq revealed that iron overload affected several signaling pathways,including Ras signaling pathway,PPAR signaling pathway,HIF-1signaling pathway,c AMP signaling pathway,MAPK signaling pathway,and PI3 K AKT signaling pathway in gastric cancer cells HGC-27.It was verified that iron overload promoted the expression of p-AKT and p-PI3 K in the PI3K-AKT pathway by Westen Blot.2.Compared with normal tissues,the TFRC and DMT1 genes were upregulated in gastric cancer tissues,and immunohistochemistry also showed that TFRC and DMT1 proteins were highly expressed in gastric cancer tissues.Consensus clustering was constructed with three subtypes of iron metabolism related molecules,named IMcluster A,IMcluster B,IMcluster C.Overall survival differed significantly between patients in the three subtypes,with IMcluster B patients having a more favorable survival advantage.Molecular biological characterization revealed high activation of DNA repair related pathways in IMcluster B.Whereas signaling pathways associated with seniority and mechanistic activation were highly activated in imclusterc.While in IMclusterc C signaling pathways associated with cancer and stromal activation are highly activated.Tumor microenvironment features revealed that IMcluster B was infiltrated with a large number of innate and adaptive immune cells.A small proportion of innate and adaptive immune cells and a large number of stromal cells were infiltrated in IMcluster C.Principal component analysis was used to construct an iron metabolism related gene signature score for patients with gastric cancer,named IMscore.Patients were divided into high and low scoring groups by choosing the optimal cut-off value,in which a high IMscore confers a more significant survival advantage.Most high IMscore patients belong to IMcluster B and most low IMscore patients belong to IMcluster C.There were significant differences in T、N stage between high and low imscore groups in clinicopathological features.Finally we found that the expression level of PD-L1 was higher in the high IMscore group,tumor mutation burden was positively correlated with IMscore,and higher imscore in patients with high microsatellite instabilit.3.There were significant differences in overall survival,tumor pathological grade as well as T stage between high and low risk groups.The time-dependent receiver operating characteristic curve showed that the AUC values of the model for predicting 1,3,and 5 years were 0.704,0.717,0.853,demonstrating the excellent accuracy of the prognostic model.Molecular features showed that cancer-related signaling pathways and stromal activation pathways were highly activated in the high-risk group,whereas DNA repair related pathways were highly activated in the low-risk group.The tumor microenvironment characteristics showed substantial stromal cell infiltration in the high-risk group,while a small proportion of innate and adaptive immune cells were infiltrated in the low-risk group.Decision curve analysis showed that the constructed nomogram had excellent predictive ability.Conclusion:1.The proliferation,migration and invasion abilities of gastric cancer cells were promoted by iron overload,and the effects on invasion and migration abilities may be mediated by affecting the PI3K-AKT signaling pathway.2.The iron metabolism pattern was altered in gastric cancer tissues,which exhibited“iron requiring” characteristics.3.Alterations in iron metabolism patterns in gastric cancer affect signaling pathways related to DNA repair,matrix activation,immune activation,and cancer related signaling pathways.Alterations in iron metabolism have some effects on remodeling of the tumor microenvironment in gastric cancer,mainly altering the infiltrative abundance of immune and stromal cells.4.The IMscore for patients with gastric cancer is able to provide a predictive probability for immunotherapy benefit.5.The prognostic model and nomogram associated with iron overload better predict patients’ 1-,3-,and 5-year survival. |
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