Study On The Antitumor Effect Of Formononetin Targeting USP5 Protein

Due to high recurrence rate and high metastasis rate,the mortality rate of hepatocellular carcinoma(HCC)has been high.Epithelial mesenchymal transition(EMT)plays an important role in the malignant progression of HCC.As an important transcription factor in EMT,SLUG promote EMT in tumor cells by inhibiting the expression of E-cadherin.Therefore,SLUG and its interacting proteins can be used as potential targets for developing anti-HCC drug.In this study,the interaction and colocation between USP5 and SLUG were found and comfirmed by affinity purification and LC-MS/MS,immunoprecipitation,immunofluorescence and ultra-high resolution experiment.The results of cycloheximide tracing experiment and in-vitro deubiquitination assay showed that USP5 stabilize SLUG through its activity of deubiquitinase.Further experiments indicated that knocking down USP5 inhibit EMT of HCC cells.Clinical data analysis showed that the transcription levels of USP5 and SLUG in HCC tissues were significantly higher than those in normal tissues.Moreover the expression level of both was closely related to histological grade and prognosis.In addition,the expression of USP5 was also correlated with the expression level of EMT marker,relevant transcription factors and metastasis related proteins.Therefore,USP5 may become a potential target for the development of anti-HCC drugs.Through screening traditional Chinese medicine library,we found that formononetin(FN)binding specifically to USP5.Further study showed that FN inhibit USP5 deubiquitinating SLUG.In vivo and in vitro pharmacodynamics experiments results indicated that FN inhibit the migration and invasion of HCC cells,and inhibit tumor growth and lung metastasis in tumor-bearing mice.This study revealed the critical role of USP5 in deubiquitinating SLUG and malignant progression of HCC,and identified FN as a novel anti-tumor compound of HCC.