The Deubiquitylase USP5 Regulates Ferroptosis Via Stabilization Of LSH In Hepatocellular Carcinoma

Background: Due to the high occurrence of recurrence and metastasis,hepatocellular carcinoma ranks the third among all cancers in terms of mortality.However,the specific mechanism of HCC has not yet been elucidated.Ferroptosis is a metabolism-related and regulated form of cell death,and its dysfunction is closely related to a variety of human diseases including cancer.Exploring the specific regulatory mechanism of ferroptosis of HCC cells is significant to the clinical diagnosis and treatment of HCC.Epigenetic regulators play key roles in the regulation of ferroptosis of cancer cells.Preliminary studies of our research group have shown that the chromatin remodeling protein LSH can promote the tumorigenesis of lung cancer and leukemia by inhibiting ferroptosis,but its role in regulating ferroptosis of HCC cells is still unclear.Deubiquitinases can remove ubiquitin from protein substrates which are essential in the regulation of cancer progression,and are closely related to the occurrence and development of cancer.A number of studies have shown that LSH is highly expressed in various cancers and is positively correlated with poor prognosis of patients,but the molecular mechanism of its high expression in cancer is rarely studied.Object: To explore the molecular mechanism of post-translational modification of LSH by deubiquitinase,thereby regulating ferroptosis in HCC cells and affecting the progression of HCC.Methods: First,mass spectrometry in 293 T cells stably expressing LSH were conducted to identify LSH-interacting deubiquitinase USP5.Then,correlation analysis,immunofluorescence and Co-IP were used to explore whether there was an interaction between USP5 and LSH.Subsequently,HCC cell lines stably overexpressing or knocking out USP5 were constructed.And these cell lines were treated with CHX or MG132,and in vivo deubiquitination experiments were also performed in these cell lines to explore whether USP5 could deubiquitinate LSH.The expression levels of USP5 and LSH in HCC and their effects on the prognosis of liver cancer patients were further analyzed by the bioinformatics analysis,western blot and Immunohistochemistry.Then,the effect of USP5 on the proliferation,migration,colony formation and tumor growth of HCC was explored through a series of biological function experiments,and the role of USP5 in regulating ferroptosis was investigated by morphological observation,cell survival ratio analysis,lipid ROS and total GSH detection using HCC cell lines that stably overexpressed or knocked out USP5.In addition,the biological function experiments and the detection of surrogate markers for ferroptosis were performed in HCC cell lines knocked out of USP5 with LSH rescued to explore whether USP5 could affect the progression of HCC and the ferroptosis of HCC cells by regulating LSH.Finally,the USP5 inhibitor Degrasyn was used to detect the effect of Degrasyn on the malignant progression of HCC and the ferroptosis of HCC cells by in vivo deubiquitination assay,biological function experiments and flow cytometry experiments.Results:(1)USP5 interacts with and stabilizes LSH protein through deubiquitination.Both USP5 and LSH are highly expressed in HCC and negatively correlated with patient prognosis.(2)USP5 can promote the proliferation,migration,colony formation and tumor growth of HCC by stabilizing LSH.USP5 can inhibit the ferroptosis of HCC cells by stabilizing LSH.Degrasyn promotes ferroptosis to inhibit tumor progression via targeting of USP5.Conclusion: USP5 could interact with LSH and stabilize LSH in a deubiquitylation activity-dependent manner,thereby inhibiting ferroptosis of HCC cells and promoting the progression of HCC.Meanwhile,the USP5 inhibitor Degrasyn can promote ferroptosis of HCC cells to inhibit HCC progression,suggesting that USP5 may become a new target for HCC therapy.