The Mechanism Of COMMD10 In Increasing Ferroptosis And Radiosensitivity Via Inhibition Of HIF-1α/CP Loop In Hepatocellular Carcinoma

Backgroud and purposeCopper is an important trace element in the body,and its imbalance of homeostasis is closely related to the occurrence and development of hepatocellular carcinoma(HCC).Radiation resistance is the main reason for the failure of radiotherapy for HCC.Serum copper is an effective indicator to monitor the efficacy of radiotherapy,indicating that copper or copper metabolism-related proteins play an important role in the radiosensitivity of HCC,but the specific mechanism is still unclear.The purpose of this study was to investigate the mechanism of copper or copper metabolism-related proteins in the regulation of radiosensitivity,so as to provide a basis for the search for biomarkers and radiosensitization targets for the effective prediction of radiosensitivity of HCC.Methods1.Real-time quantitative PCR(QPCR)was used to detect the expression of COMMD family in HCC cells with or without radiation;2.Construct a radiation-resistant HCC strain and detect the expression of COMMD10 by QPCR;3.Immunohistochemical detection of COMMD10 expression in radiosensitive and resistant tissues;4.Lentivirus infection was used to construct gene knockdown/overexpression cell lines;5.The radiosensitivity of HCC cells was detected by plate cloning and CCK8 assay;6.Protein spectrum combined with death pathway inhibitors was applied to screen radiation-induced tumor cell death;7.Flow cytometry was used to detect cell lipid reactive oxygen species(lipid ROS);8.The levels of malondialdehyde(MDA),glutathione(GSH)and Cu2+ were detected by microplate reader.9.Laser confocal was applied to detect Fe2+ fluorescent probe;10.Bioinformatics was employed to investigate the radiosensitivity index,protein interaction network and downstream enrichment pathway of COMMD10;11.The interaction between COMMD10 and HIF-1α was detected by immunoprecipitation;12.Western blot was used to examine HIF-1α protein half-life,ubiquitination,CP and SLC7A11 expression;13.The subcutaneous tumor radiation model was constructed for in vivo experiment.Resutls1.Cu2+concentration in HCC cells was associated with radiation resistance.The expression of copper metabolism-related protein COMMD10 in HCC cells after radiation showed a dose-dependent decrease,and its expression in radiation-resistant strains was significantly lower than that in parental cells.It was highly expressed in radiation-sensitive tissues and poorly expressed in radiation-resistant tissues.2.Low expression of COMMD10 induced radiation resistance of HCC cells,while overexpression of COMMD10 increased the radiosensitivity of HCC cells.Tumor radiosensitivity index analysis showed that COMMD10 plays a role of radiosensitization in different tumors.3.In vivo and in vitro experiments showed that:COMMD10 promoted irradiated HCC cells to generate lipid ROS and MDA,and increased the ferroptosis and radiosensitivity.4.Under both normoxic and hypoxic conditions,COMMD10 depletion could increase the stability of HIF-1α by increasing the intracellular Cu2+ and prolong the half-life of HIF-la and inhibit its ubiquitination degradation.Co-immunoprecipitation results confirmed that the N-terminal of COMMD10 directly bound to HIF-1α,and inhibited HIF-1α nuclear translocation and its downstream target genes CP and SLC7A11 expression.Subcutaneous tumor radiation model experiment results showed that COMMD 10-knockdown cells derived tumors was significantly larger than control group after radiation,and had decreased lipid ROS as well as increased Ki67 proliferation activity.HIF-1α inhibitors suppressed COMMD 10-knockdown mediated radiation resistance.5.CP overexpression increased HIF-la by reducing Fe2+in HCC cells after radiation,forming a positive feedback loop of HIF-lα/CP.COMMD 10 decreased GSH while increased Fe2+,lipid ROS,MDA,ferroptosis and radiation sensitivity by inhibiting HIF-1α/CP loop in HCC cells.ConclusionsOur findings suggest that COMMD 10 increases radiosensitivity of HCC cells by inhibiting HIF-1α/CP positive loop.COMMD10 is a potential biomarker of responders for radiotherapy and a potential radiation sensitizer in HCC.Innovation1.COMMD10 was firstly identified as a new intersecting gene that regulates copper and iron metabolism in irradiation treated HCC cells.2.A new mechanism was revealed for the first time:COMMD10 increased ferroptosis and irradiation sensitivity via inhibition of HIF-1α/CP loop in HCC.3.A new target was proposed for the first time:COMMD10 can be used as a potential target for the prediction of radiation efficacy and radiosensitization of HCC.