USP27 Regulates Proliferation And Migration Of Hepatocellular Carcinoma Cells Via Catalyzing SETD3 Deubiquitination

In recent decades,cancer has become one of the most important causes threatening human health,and hepatocellular carcinoma is the most common malignant tumor.With the continuous improvement of medical technology and methods,the treatment of hepatocellular carcinoma is becoming more and more perfect,but the research on the mechanism of the occurrence,development and metastasis of hepatocellular carcinoma has not made breakthrough progress.It is great significance to study the biological regulation mechanism of hepatocellular carcinoma so as to obtain effective treatment methods.Ubiquitination and deubiquitination of proteins belong to post-translational modifications in epigenetics,which have the functions of regulating cell signal transduction,DNA damage repair,RNA splicing and so on.The ubiquitination and deubiquitination of proteins have been proved to regulate the function of related proteins by changing their stability and protein-protein interaction,thus regulating the occurrence,development and metastasis of tumors.USP27 is a deubiquitinase that exists in humans and higher animals,and its structure consists of two parts,N-terminal containing zinc finger structure and C-terminal containing specific ubiquitin peptide functional domain.Studies have shown that USP27 is abnormally expressed in the liver,and affects the abnormal expression of cyclin through deubiquitination function,thus affecting the occurrence and development of tumors.However,the molecular mechanism of USP27 in the occurrence and development of hepatocellular carcinoma has been rarely reported,and the mechanism of USP27 in hepatocellular carcinoma is still unclear.Therefore,the study of USP27 and its downstream target proteins is of great scientific significance for understanding the occurrence and development of hepatocellular carcinoma.In this study,we investigated the molecular mechanism by which USP27 regulates the occurrence and development of HCC by affecting the expression of SETD3.The main results are as follows:1.USP27 interacts with USP27 and co-localized in cellsThe interaction between USP27 and SETD3 was demonstrated by co-immunoprecipitation experiments,and the interaction between USP27 and SETD3 was mainly mediated by the C-terminal of USP27 containing UCH domain.Immunofluorescence assay showed that USP27 and SETD3 were co-localized in human hepatoma Hep3 B cells.2.USP27 deubiquitinates SETD3 and stabilizes SETD3 expressionAfter determining the interaction between USP27 and SETD3,we further verified the effect of USP27 and SETD3 interaction on SETD3.Using the ubiquitin degradation experiment,we found that USP27 could deubiquitinate SETD3 and stabilize SETD3expression;in the human hepatoma Hep3 B cell line knocked down by USP27,the expression level of SETD3 decreased.3.Knockdown of USP27 inhibits the proliferation and migration ability of hepatoma cellsIn order to study the effects of USP27 and SETD3 on the occurrence and development of hepatocellular carcinoma,we constructed stable knockdown cell lines of USP27 and SETD3 in human hepatocellular carcinoma cell Hep3 B,and detected the proliferation and migration ability of each cell line by clonogenic assay and cell scratch assay.The results showed that both USP27 knockdown and SETD3 knockdown could inhibit the proliferation and migration ability of human hepatoma cell Hep3 B.4.Expression level and survival analysis of USP27 and SETD3 in hepatocellular carcinomaLiver cancer tissues were stained by immunohistochemistry,and the results showed that USP27 and SETD3 were highly expressed in liver cancer tissues compared with normal liver tissues.The protein level analysis of 10 groups of patients’ clinical samples showed that both USP27 and SETD3 were highly expressed in cancer tissues and low expressed in paracancerous tissues.The clinical expression of USP27 and SETD3 was analyzed by GEPIA database with liver cancer.Relationship between patient survival,patients with low expression of USP27 and SETD3 had longer survival than those with high expression of USP27 and SETD3.