Mechanism Of RNF8 On Iron Metabolism And Liver Ferroptosis By Regulating Steap3

Objective: Ubiquitin ligase RNF8 plays an important role in DNA damage repair and cell cycle regulation,and our recent study found that it also plays a role in ferroptosis.Ferroptosis is an iron-dependent death pathway that catalyzes lipid peroxidation of highly expressed unsaturated fatty acids in cell membranes under the action of divalent iron or ester oxygenase,leading to cell death.In recent years,more and more evidence indicates that ubiquitination plays an important role in regulating ferroptosis,but the specific regulatory mechanism remains unclear.Our study discusses the specific regulatory mechanisms of RNF8 on ubiquitination of Steap3,as well as its role in iron metabolism and liver ferroptosis.Methods: The effects of RNF8 gene deletion on iron metabolism in mice were investigated by detecting serum iron,total iron-binding ability,and tissue iron.The effects of RNF8 gene deletion on liver ferroptosis in mice were investigated by detecting liver divalent iron,MDA,GSH,and other ferroptosis-related phenotypes.Lentivirus-infected HL7702 cell lines were used to construct RNF8 knockdown and overexpression stable strains,and ferriamine citrate（FAC）was used to induce the establishment of the high iron model in cells.Firstly,the optimal concentration of FAC was determined by CCK-8 and RTCA experiments,and DFO and FER-1 ferroptosis inhibitors were used to verify the ferroptosis of cells.Furthermore,iron content in cells was measured and Prussian blue staining was used to explore the deposition of iron in cells;MDA,GSH,ALT,and AST were detected to explore the degree of peroxide damage in cells;FTH1 and GPX4 protein expression levels in cells were detected by Western Blot to explore the antioxidant capacity of cells.Mito Tracker live-cell staining was used to investigate the function of mitochondria and the effect of RNF8 knockdown on ferroptosis in human liver cells.Through a combination of proteomics and modification omics,we identified the downstream protein Steap3,which is associated with iron metabolism and ferroptosis.Western Blot was used to verify whether there was a correlation between the protein expression levels of RNF8 and Steap3.Co-IP and IF experiments were used to further verify the interaction between the two proteins,and Co-IP experiment was used to verify the ubiquitination pathway of Steap3.Results: Compared with WT mice,RNF8 gene deletion mice showed lower serum iron and total iron-binding ability,no statistical difference in hemoglobin and serum iron divalent,increased iron deposition in the liver,and no statistical difference in other tissues and organs.After the high iron diet,compared with WT mice,the binding force of serum iron and total iron was increased in RNF8-deficient mice,Prussian blue staining showed increased iron deposition in the liver and duodenum,and decreased iron deposition in the spleen.ALT and AST in serum increased,and HE staining showed increased necrosis area in the liver.In liver tests,iron divalent and MDA increased,GSH decreased,FTH1 and GPX4 protein expression levels decreased,Masson staining and Sirius red staining showed increased liver fibrosis.After FAC induction of HL7702 cells,compared with LV-con313 group,iron content and MDA in LV-RNF8 i group increased,GSH decreased,ALT and AST increased,FTH1 and GPX4 protein expression levels decreased,Prussian blue staining showed increased iron deposition in cells.Mito Tracker living cells stained with weakened mitochondrial function.In the HL7702 cell line,RNF8 was negatively correlated with the protein expression level of Steap3.RNF8 was an interaction protein with Steap3 and could modify Steap3 in the form of a K48 ubiquitin chain.Conclusion: Iron metabolism in RNF8 knockout mice was abnormal.After high iron induction,loss of RNF8 resulted in increased ferroptosis in the liver,increased oxidative damage,weakened antioxidant capacity,massive necrosis and fibrosis in liver tissues,and RNF8 knockdown resulted in weakened mitochondrial function.In this study,we found that RNF8 and Steap3 are interacting proteins,and RNF8 is negatively correlated with the protein expression level of Steap3 and can modify Steap3 in the form of K48 ubiquitination chain,thereby regulating iron metabolism and ferroptosis.In conclusion,this study reveals the ubiquitination pathway of Steap3 and provides new insights into the role of RNF8 in regulating ferroptosis ubiquitination.