Research Of USP22 Promoting Hypoxia-induced Hepatocellular Carcinoma Stemness

BackgroundHepatocellular carcinoma(HCC)is one of the most common malignant tumors with poor prognosis in China.Due to the high prevalence of hepatitis B,almost 90% of HCC are HBV-related.Surgical resection,transcatheter arterial chemoembolization(TACE),radiofrequency ablation(RFA)and other auxiliary treatment methods are helpful for patients,but have no significant effect on advanced HCC patients.So it is important to explore the pathogenesis of HCC and find new targets for treatment to improve the prognosis of HCC patients.Because of unrestricted proliferation and abnormal angiogenesis,hypoxia is common in solid tumors.HIF1α is a subunit of HIF1,becomes stable in hypoxia and regulates the expression of genes related to cancer stemness.USP22 was identified as a cancer stem cell marker in previous studies,and our team found that USP22 could regulate the resistance of HCC cells to cytotoxic drugs by MRP1.TP53 is one of the most important tumor suppressor genes.Mutation of TP53 is common in patients with HBV-related HCC.In this study,it was found that under hypoxia condition,USP22 could stabilize HIF1α and HIF1α could promote the transcription of USP22 in the absence of TP53.The purpose of this study is to investigate the effect of USP22/HIF1α on cancer stemness,the mechanism of USP22 regulating HIF1α and the clinical significance of USP22/HIF1α as a prognosis marker and therapeutic target.Aims1.To identify the role of USP22 in promoting the HCC stemness under hypoxia condition.2.To explore the mechanism of USP22 promoting the HCC stemness by HIF1α in HCC cells under hypoxia condition.3.To explore the clinical significance of USP22 in HCC patients and whether it can serve as a target for HCC treatment.Methods1.Lentivirus was used to construct USP22 silent or overexpressed HCC cell lines.RNA-seq was used to prove that USP22 could promote the expression of stemness-related genes under hypoxia condition.Cell counting,sphere formation,migration,drug resistance to sorafenib,stemness related surface markers,and in vivo extreme limiting dilution assays were used to evaluate the change of stemness.2.Western blot and Luciferase reporter assay were used to detect the influence of USP22 on HIF1α protein level and transcriptional activity.The mutant plasmids of USP22 and HIF1α were constructed.By using co-immunoprecipitation and immunofluorescence assays,the binding sites of USP22 and HIF1α were identified,as well as the binding location and the mechanism USP22 regulates HIF1α.3.The correlation of USP22 and HIF1α expression from 262 HCC patients was determined and analyzed by immunohistochemistry.325 patients from TCGA database were included to evaluate the relation between m RNA of USP22 and HIF1α,mutation condition of TP53 and the survival time.Nanoparticles targeting USP22 and small molecule inhibitor sorafenib were used to treat the tumor in vivo.The therapeutic effect and weight of each group were observed after 5 weeks.The expressions of USP22 and HIF1α in tumors were analyzed by immunohistochemistry.Results1.Under the hypoxia condition,in vitro experiments,USP22 could promote the proliferation,sphere formation,migration,drug resistance to sorafenib and the expression of stemness-related surface markers of HCC cells;In vivo experiments,USP22 promoted the tumorigenesis of HCC cells.2.Downregulation of USP22 in HCC cell lines decreased the expression and transcriptional activity of HIF1α,while overexpression of USP22 promoted the expression and transcriptional activity of HIF1α.The C19 peptidase domain of USP22 bond with HIF1α and the co-localization were in the nucleus.Based on the mutation of TP53,HIF1α promoted the transcription of USP22.Knockdown of TP53 in wild-type cells restored the transcriptional promotion of HIF1αon USP22.3.Patients with high expression of USP22 and HIF1α,TP53 mutation had a very poor clinical prognosis;FLPP-sh USP22,a nanoparticle targeting USP22,inhibited the tumor growth of TP53 wild-type cells in vivo.And combined with small molecule inhibitor sorafenib,FLPP-sh USP22 suppressed the tumor growth of TP53 mutant cells.Conclusions:1.USP22 promotes the cancer stemness of HCC cells under hypoxia condition.2.USP22 inhibits the ubiquitination of HIF1α so as to stabilize the protein and increase its transcriptional activity.USP22 binds with HIF1α in the nucleus through the C19 peptidase domain.Based on the TP53 mutation,HIF1α expression promotes the expression of USP22 as a transcription factor.3.USP22,HIF1α combined with TP53 mutation are potential prognostic markers of HCC patients.Nanoparticles targeting USP22 showed good anti-tumor effects.