Function And Molecular Mechanism Of USP27 And USP39 In Tumorigensis Of Hepatocellular Carcinoma

Liver cancer is one of the most commonly diagnosed cancer in China and has high mortality which account for about 50% of the world.Liver cancer usually originates from hepatitis lesions.Hepatitis B virus positively regulates many substrates and kinases in multiple signaling pathways in liver cells,which promotes liver abnormalities and then leads to cancer.Hepatocellular carcinoma(HCC)is the most common type of primary liver cancer and involves multi-step regulation of genes and factors.HCC has many remarkable characteristics of easy relapse,high metastasis and poor prognosis.Therefore,it is crucial to clarify the molecular mechanisms of liver cancer development to find effective biological therapeutic targets.Specific genes expression especially transcription and translation are involved in the formation and development of HCC.Post-translational modification including phosphorylation,ubiquitination,and acetylation is a common way to regulate protein expression.This study mainly explores the specific functions and regulatory mechanisms of ubiquitinating-and acetylating-modification in HCC.The research has two parts.Part 1: USP27-mediated Cyclin E stabilization drives cell cycle progression and hepatocellular tumorigenesisCancer is of a multi-gene regulatory and systemic disease that involves the activation of proto-oncogenes and the inactivation of tumor suppressor genes and ultimately leading to abnormalities of cell cycle during the process of origination,progression and termination.Cancer cells always contain a rapid growth and reduced apoptosis.Many studies have indicated that the continuous high expression of Cyclin E leads to cell cycle abnormalities and promotes the development of various cancer.The abnormal expression of Cyclin E in HCC is significantly associated with poor prognosis.There have many studies on Cyclin E modification and regulation,while its specific deubiquitinase has not been reported,and its molecular mechanism in the process of hepatocellular carcinoma remains to be studied.Therefore,this study mainly to find Cyclin E deubiquitinating enzymes and the specific functions and mechanisms of regulating Cyclin E ubiquitination and stability,as well as the mechanism of deubiquitinaes regulating liver cancer progression by mediating Cyclin E.The main methods and results in research are as follows:(1)USP27 negatively regulates ubiquitination of Cyclin E to promote its stabilityDUB library screening confirmed that USP27 is a deubiquitinating enzyme of Cyclin E.USP27 and Cyclin E had specific interaction and co-localize in Hep3 B cells.Overexpression of USP27 inhibited the ubiquitination and degradation of Cyclin E and prolonged its half-life.Knockdown of USP27 promoted ubiquitination and degradation of Cyclin E protein and shortened its half-life.After the active site of USP27 deubiquitinase was deleted,there was no significant change in the ubiquitination of Cyclin E.Cyclin E expression was significantly down-regulated in USP27-knockdown Hep3 B cells,however MG132 treatment could rescue the USP27 knockdown mediated degradation of Cyclin E protein.(2)USP27 mediated Cyclin E to promote the development of hepatocellular carcinomaKnockdown expression of USP27 in Hep3 B and MHCC97 H cells blocked G1/S in the cell cycle and inhibited cell proliferation and colony formation.Reintroducing Cyclin E into USP27-depleted cells restored the malignant phenotype.Conversely,overexpression of USP27 promoted cell proliferation and colony formation compared with the vector control.Knockdown of USP27 inhibited subcutaneous tumor formation in nude mice.(3)5-FU negatively regulates the expression of USP27 and inhibits the proliferation of liver cancer cellsUSP27 and Cyclin E expression were downregulated by 5-fluorouracil(5-FU)treatment.The protein expression of USP27 decreased in a dose-dependent manner(Fig.7b)or changed with time increasing following 5-FU treatment.Notably,knockdown of USP27 expression together with 5-FU treatment greatly reduced the number of cells in G2/M phase.Furthermore,cell proliferation was also remarkably reduced and cell apoptosis was induced in the combination of USP27 knockdown and 5-FU treatment.Our discovery demonstrates that USP27 promotes the growth,migration,and tumor growth of hepatocellular carcinoma cells through Cyclin E,and provides a theoretical basis by regarding USP27/Cyclin E as a potential therapeutic target for liver cancer.Part 2: An NAD+-dependent deacetylase SIRT7 promotes HCC development through deacetylation of USP39Acetylation is one of post-translational modification of proteins.More than 2,000 proteins have been acetylated.Acetylation is a widely modification of protein in cells which involves in apoptosis,DNA transcription and repair,chromatin recombination and proteins stability and other processes.USP39,as a deubiquitinating enzyme,was responsible for the formation of spliceosome.It mainly recruites ribonucleoproteins U4/U6 U5 to form triribonucleoproteins,then promotes the formation of stable and integral spliceosome.It was reported that USP39 was predicted to have many modification sites of phosphorylation,acetylation and ubiquitination by mass spectrometry,but lack of experiment support.Moreover,the high expression of USP39 in HCC is closely related to the poor prognosis.Therefore,we analyzed the USP39 acetylation regulation to explore the function and molecular mechanism of USP39 in HCC development.The main research methods and results are as follows:(1)SIRT7 negatively regulates USP39 acetylationMass spectrometry combined with co-immunoprecipitation confirmed the specific interaction between USP39 and SIRT7.The overexpression of USP39 did not affect the ubiquitination level of SIRT7 protein,but knockdown of SIRT7 can significantly promote the acetylation of USP39 protein.Overexpression of SIRT7 significantly inhibited the acetylation of USP39 protein.After the active site of SIRT7 protease was deleted,there is no change in regulation of USP39 acetylation.(2)SIRT7 mediates USP39 stabilityUbiquitination of USP39 was increased in SIRT7-depleted cells which reduced USP39 expression in the nuclear.The expression of USP39 protein was elevated in SIRT7 knockdown cells treated with MG132.Knockdown of VHL significantly increased the expression of USP39 protein in SIRT7-knockdown cells.Lysine 508 act as the main acetylation site of USP39 protein.Overexpression of SIRT7 significantly reduced the acetylation level of USP39,but had no effect on the acetylation of USP39 K508 R mutants.(3)SIRT7-mediated deacetylation of USP39 promotes cell growth and tumorigenesis of HCCSIRT7-depleted cells had significantly decreased the ability of proliferation and colony formation,while reintroducing USP39 WT or KR into USP39-knockdown cells restored the malignant phenotype.However,co-expressing SIRT7 and USP39 WT could furtherly promote the proliferation and colony formation.Knockout of SIRT7 significantly inhibited subcutaneous tumor formation in nude mice,but reintroducing USP39 WT or KR restored tumor growth.The results of this study indicats that SIRT7 mediates the acetylation level of USP39 which regulates the stability of USP39 and participates in the regulation of liver cancer cell growth,proliferation and tumor formation.Inhibition of SIRT7/USP39 will provide a new strategy and method for clinical treatment of HCC.