The Role And Mechanism Of SCARA5 Combined With Ferritin Induced Ferroptosis In Esophageal Cancer

Objective:Esophageal squamous cell carcinoma(ESCC)is one of the deadliest cancers in the world with an extremely poor prognosis.Scavenger receptor protein 5(SCARA5),as a membrane protein,plays an important role as a tumor suppressor gene in various cancers.Therefore,this study aimed to investigate the function of SCARA5 gene in esophageal squamous cell carcinoma and explore the potential mechanism of action.Methods:First,whole transcriptome sequencing of 6 pairs of ESCC tissues and adjacent tissues was performed to screen out the low-expressed gene SCARA5 in esophageal squamous cell carcinoma.RT-qPCR and Western blot were used to analyze the expression difference of SCARA5 in normal esophageal epithelial cells and esophageal squamous cell carcinoma cells,and immunohistochemical staining was used to analyze the expression difference of SCARA5 in ESCC tissue and adjacent tissue,and the expression difference and prognosis analysis of SCARA5 were analyzed in combination with the database.Then,lentivirus was used to construct stable cell lines overexpressing SCARA5.Flow cytometry was used to analyze cell cycle changes,and wound healing and Transwell experiments were used to analyze cell migration and invasion abilities.Use CCK-8 assay to detect cell proliferation activity and use cytostatic to explore changes in cell proliferation activity.Then,the morphological changes of cell mitochondria were observed by transmission electron microscope,and the concentration of total reactive oxygen species and lipid reactive oxygen species in cells was detected by flow cytometry.At the same time,the metabolic changes of intracellular lipid reactive oxygen metabolite malondialdehyde and intracellular glutathione peroxidase substrate glutathione were detected.Further detection of intracellular iron metabolism changes by immunofluorescence and flow cytometry experiments.The binding of SCARA5 to ferritin was detected by co-immunoprecipitation assay,and the changes of intracellular ferroptosis-related proteins were analyzed by Western blot.Finally,a nude mouse subcutaneous tumor model was constructed to observe the effect of overexpression of SCARA5 on tumor growth.Results:The whole transcriptome sequencing results of 6 pairs of ESCC tissues and adjacent tissues showed that the SCARA5 was low expression in ESCC tissues;RT-qPCR and Western blot analysis showed that SCARA5 was significantly lower in esophageal squamous cell carcinoma cells than in normal esophageal epithelial cells,especially TE-1 and KYSE150;the results of immunohistochemical analysis showed that SCARA5 was significantly lower expressed in ESCC compared with adjacent tissues;the expression of SCARA5 in ESCC was significantly lower than that in normal tissues in the GEPIA database;the prognostic analysis of esophageal cancer patients in the TCGA database showed that patients with low SCARA5 expression had a worse prognosis.The results of RT-qPCR and Western blot verification showed that TE-1 and KYSE150 stable cell lines successfully overexpressed SCARA5,Flow cytometry detection of cell cycle found that overexpression of SCARA5 arrests TE-1 and KYSE150 cell cycle in G0/G1 phase;wound healing and Transwell assays showed that overexpression of SCARA5 significantly inhibited the migration and invasion of esophageal squamous cell carcinoma cells.CCK-8 experiments showed that overexpression of SCARA5 significantly inhibited the proliferation activity of esophageal squamous cell carcinoma cells;after treatment of cells with necrosis inhibitor Necrostatin-1,apoptosis inhibitor ZVAD-FMK,and autophagy inhibitor 3-MA,it was found that the activity of esophageal squamous cell carcinoma cells in overexpression SCARA5 group was still lower than that of cells in control group;after the cells were treated with the ferroptosis inhibitor Ferrostatin-1,compared with the control group,the cell activity of the overexpressing SCARA5 group increased;after the cells were treated with the ferreptosis inducer Erastin,compared with the control group,the lower cell viability in the overexpressing SCARA5 group.Transmission electron microscopy experiments found that compared with the negative control group,the mitochondrial volume decreased,the mitochondrial cristae decreased,the mitochondrial membrane density increased in overexpression SCARA5 groups,and appearance the typical ferroptosis morphological changes;flow cytometry was used to detect the total intracellular reactive oxygen species and lipid reactive oxygen species.It was found that overexpression of SCARA5 resulted in an increase in the total intracellular reactive oxygen species and lipid reactive oxygen species.At the same time,the intracellular lipid reactive oxygen metabolite MDA increased,and the substrate glutathione of glutathione peroxidase decreased.Immunofluorescence experiments and flow cytometry experiments were used to detect changes in intracellular iron metabolism.It was found that overexpression of SCARA5 caused a decrease in intracellular green fluorescence,indicating an increase in intracellular iron ions.Further co-immunoprecipitation experiments confirmed that SCARA5 binds to ferritin.Western blot experiments showed that overexpression of SCARA5 increased the expression of light chain ferritin and heavy chain ferritin but did not affect the expression of transferrin receptors.Finally,subcutaneous tumor experiments in nude mice showed that overexprcssion of SCARA5 inhibited the growth of subcutaneous tumors in nude mice.Conclusion:The expression of SCARAR is significantly lower in esophageal squamous cell carcinoma tissues and esophageal squamous cell carcinoma cells,and the prognosis of patients with low expression of SCARA5 is worse.Overexpression of SCARA5 arrests the cell cycle ofesophageal squamous cell in G0 phase,and overexpression of SCARA5 inhibits the migration and invasion of esophageal squamous cell carcinoma cells.Overexpression of SCARA5 induces ferroptosis in esophageal squamous cell carcinoma cells,resulting in decreased cell proliferation activity.SCARA5 increases the accumulation of intracellular reactive oxygen species and the increase of iron ions by binding to ferritin,thereby inducing ferroptosis in esophageal squamous cell carcinoma cells.