Exploring The Expression,Function And Mechanism Of Deubiquitinating Enzyme USP19 In Hepatocellular Carcinoma

【Background】HCC ranked sixth among common malignancy tumors worldwide,accounting for75-85% of all primary liver cancers.There were 410,038,new cases of HCC in China,resulting in 391,152 deaths according to WHO 2020.Asia and Africa exhibit the highest incidence rates of HCC worldwide,with China accounting for approximately half of all HCC patients globally.The onset of HCC is hidden,and many patients are already in the middle and late stages of treatment.Many patients can only take palliative care such as TACE and molecular targeted therapy,resulting in high mortality.Therefore,in-depth study of the molecular biology underlying the pathogenesis of HCC,along with the pursuit of developing molecular therapeutic targets,holds significance in enhancing the prognostic outcomes for HCC patients.HCC is difficult to treat due to its complex molecular mechanisms of occurrence and progression,which are governed by multiple signaling cascades such as PI3K-AKT-m TOR,Wnt/β-catenin,JAK/STAT,Hippo,Hedgehog,and Notch.The Hippo pathway,in particular,exhibits some distinctive features.It is a highly conserved signaling pathway that plays a crucial role in restricting organ size,tissue regeneration,and cell proliferation.YAP is one of the major factors of the Hippo signaling.However,the mechanism of abnormal YAP activation in HCC has not been well elucidated.DUBs exert a crucial influence on the oncogenesis of malignancies.There are multiple substrate proteins that can be modified by deubiquitination in the Hippo pathway.However,studies on Hippo pathway mainly focus on phosphorylation,and there are limited literature on the deubiquitination modification of Hippo pathway.The regulatory role of these DUBs in Hippo pathway is still not completely clear.【Objectives】1.Identifity DUBs that can regulate HCC progression via Hippo pathway.2.To investigate the expression of deubiquitination enzyme USP19 in HCC,and explore the the clinical correlation between USP19 and HCC.3.To verify the regulatory function of USP19 in HCC.4.To elucidate the molecular mechanism of USP19 regulating HCC progression.【Methods】1.The siRNA library targeting DUBs was screened,and YAP protein was utilized as the substrate for screening through Western Blot analysis to identify the specific Dub(USP19)involved in regulating YAP protein.Experiments with CHX or MG132 were conducted to investigate whether USP19 affects YAP protein via proteasome pathway.2.The expression and prognosis of USP19 in HCC were determined through bioinformatics analysis of the TCGA database using R language software.Tumor and adjacent tissues of HCC patients were collected,along with relevant clinical information.q RT-PCR,Western Blot,and immunohistochemical experiments were employed to investigate the expression of USP19 in HCC and its correlation with clinical data.3.In HCC cell lines,the functions of USP19 on cell growth,migration,and apoptosis were validated through various in vitro experiments including plate cloning,EDU assay,CCK8 assay,wound healing assay,Transwell assay,and Annexin V-FITC/PI flow cytometry.Additionally,subcutaneous xenograft was used for investigating the HCC cells proliferation and and lung metastatic models were used for evaluating tumor metastatic ability in vivo.4.The deletion construction of USP19 and YAP were constructed to determine their interaction domains via co-immunoprecipitation experiments.We further explored the deubiquitination types of YAP using a series of ubiquitin mutants(K6,K11,K27,K29,K33,K48 and K63).Moreover,the mutant variants of YAP at its lysine sites were conducted to investigate the specific lysine sites of YAP binding to ubiquitin.【Results】1.In order to identify the DUBs that regulate YAP deubiquitylation in HCC,we screened a DUBs’ siRNA library targeting DUBs.The results showed that knockdown USP19 considerably decrease YAP protein expression.MG132 can reverse the inhibition of USP19 depletion on YAP protein expression.The result of CHX experiment indicated that USP19 knockdown decreased the stability of YAP protein.2.The in-depth analysis of the TCGA database proved that the expression of USP19 was elevated in cancer tissues,correlating significantly with an unfavorable prognosis.Both RT-q PCR and WB results demonstrated that USP19 was elevated in tumor tissues compared to adjacent non-tumor tissues.In addition,IHC findings corroborate a positive association between the expression levels of USP19 and tumor number,tumor size,and BCLC stage.3.USP19 functions as an oncogenic driver.Knockdown of USP19 effectively suppresses the proliferative and migratory capabilities of HCC cells,both in vitro and in vivo.4.USP19 interacts with WW domain of YAP via its USP domain in the cytoplasm,promoting the stabilization of YAP via reducing the K11-and K48-linked poly-ubiquitination of YAP at K76 and K90 sites.【Conclusions】In this study,we screened a DUBs’ siRNA library and reported for the first time the potential role of USP19 as a DUB involved in YAP deubiquitination and stability in HCC.USP19 interacts with WW domain of YAP via its USP domain,reducing the K11-and K48-linked poly-ubiquitination of YAP at K76 and K90 sites,promoting the stabilization of YAP in a DUB activity-dependent manner,which subsequently enhances the proliferative capacity of HCC cells in vitro and in vivo.Targeting USP19 as a YAP regulator may prove to be a feasible therapeutic strategy for HCC.