Molecular Mechanism Of USP22 Up-regulation VEGFA Gene Transcription And Its Effect In Hepatocellular Carcinoma

Objective: Primary liver cancer is predicted to be the sixth most frequently diagnosed cancer and the fourth leading cause of cancer death in 2018 worldwide.Hepatocellular carcinoma(HCC)is reported as the most common form of primary liver cancer.Most patients with HCC are diagnosed at an advanced stage.Studies on the molecular mechanism of HCC occurrence and development are of great significance for develop novel strategies to treat this disease.Vascular endothelial growth factor A(VEGFA)is a key regulator of angiogenesis,antiangiogenic therapy is considered a promising cancer treatment strategy of HCC.It is necessary to study the molecular mechanism affecting VEGFA expression in HCC.Ubiquitin specific peptidase 22(USP22),a member of the deubiquitylation enzymes(DUBs)family.A pro-oncogenic role for USP22 has been demonstrated in HCC,but the specific mechanism is still unclear.This study explored the molecular mechanism of USP22 in regulating VEGFA transcription,and its effect on cell proliferation,tumor growth and vasculogenic mimicry(VM)of HCC,providing new ideas for the exploration of HCC treatment and targets.Methods: 1.Western blot was used to detect the expression of USP22 and VEDFA proteins in HCC tissues.Pearson correlation analysis was performed to analyze the correlation between the expression of the two proteins in HCC tissues.2.The expressions of HIF-1? and VEGFA protein and m RNA were detected by Western blot and real-time PCR after silencing USP22 with si RNA.3.Protein co-immunoprecipitation was used to verify the interaction between USP22 and HIF-1? protein;Immunofluorescence staining and Confocal method were used to detect the localization of USP22 and HIF-1? protein in HCC-LM3 cells.4.After silencing USP22 with si RNA and adding CHX to inhibit protein synthesis,the expression of HIF-1? and USP22 proteins was detected by Western blot.5.Protein co-immunoprecipitation was used to detect the HIF-1? ubiquitination level after silencing USP22.6.The effect of USP22 on transcriptional activity of VEGFA promoter was detected by dual-luciferase reporter assay,and the effect of USP22 knockdown on USP22,HIF-1? and H2Bub1 recruitment in VEGFA promoter region was detected by chromatin immunoprecipitation assay.7.HCC-LM3 cell proliferation was detected by plate clone formation assay and MTS cell activity assay.8.HCC tumor growth was detected by subcutaneous tumor-bearing experiment in nude mice with HCC-LM3 cells.9.Vascular endothelial cell angiogenesis experiment and HCC-LM3 cell vasculogenic mimicry experiment were used to detect the cell angiogenesis ability.Results: 1.High levels of USP22 and VEGFA proteins were detected in HCC tissue and their expression was positively correlated.2.USP22 silencing decreased VEGFA protein and m RNA expression,it also decreased HIF-1? protein expression under hypoxia.3.An interaction between USP22 and HIF-1? protein under hypoxia,and USP22 co-localization with HIF-1? in the nucleus of HCC-LM3 cells under hypoxia.4.USP22 silencing increasing degradation rate of HIF-1? under hypoxia.5.USP22 silencing increasing the level of HIF-1? ubiquitination under hypoxia.6.Overexpression of USP22 increased the transcriptional activity of VEGFA promoter,and knockdown of USP22 increased the recruitment of H2Bub1 in VEGFA promoter region.Knockdown of USP22 decreased the recruitment of HIF-1? in VEGFA promoter region under hypoxia.7.Knockdown of USP22 inhibited the cell proliferation and clone formation ability of the HCC-LM3 cells.8.Knockdown of USP22 inhibited tumor growth in HCC-LM3 tumor-bearing mice.9.Knockdown of USP22 inhibited tube formation of vascular endothelial cell and HCC-LM3.Conclusion: 1.USP22 and VEGFA are highly expressed in HCC tissues and their expression was positively correlated.2.USP22 promoting VEGFA transcription by stabilizes HIF-1? protein expression under hypoxia.3.USP22 promoting VEGFA transcription by increases HIF-1? recruitment in the VEGFA promoter region under hypoxia.4.USP22 promotes VEGFA transcription by reducing H2 B ubiquitination in the VEGFA promoter region.5.USP22 promotes HCC cell proliferation,tumor growth and vasculogenic mimicry.