Mechanism Study On Deubiquitinase USP3 In Promoting The Proliferation Of Hepatoma Carcinoma Cells

Objective:To investigate the molecular mechanism of ubiquitin specific protease 3(USP3) in promoting the proliferation of hepatoma carcinoma cells via murine double minute 2(MDM2), which finally provides an experimental basis for USP3 as the molecular therapy target of hepatocellular carcinoma. Methods:1. Co-immunoprecipitation was used to detect the interaction between endogenous USP3 and MDM2 in HepG2 cells and HL-7702 cells, and exogenous USP3 and MDM2 in HepG2 cells.2. Western blot analysis was used to identify the type of polyubiquitination chain on MDM2 cleaved by USP3 in HepG2 cells, and while the inactive mutants of USP3, H56 A and C168 S had any effects on the deubiquitinase activity of USP3.3. Western blot analysis was used to determine whether the protein level of MDM2 could be stabilized by USP3, and whether the inactive mutants of USP3, H56 A and C168 S had any effects on this stabilization.4. Cell Counting Kit 8(CCK8) was performed to detect the effects of USP3 and the inactive mutants of USP3, H56 A and C168 S on the proliferation of HepG2 cells, and whether the presence of the MDM2 specific inhibitor Nutlin3 had any effects on the proliferation. Results:1. Endogenous and exogenous USP3 and MDM2 both had interaction in Hep G2 cells, however endogenous USP3 and MDM2 had almost no interaction in HL-7702 cells.2. USP3 could stabilize the protein level of MDM2 by cleaving the K48-type polyubiquitination chain on MDM2, while the inactive mutants of USP3, H56 A and C168 S lost this ability.3. Only USP3 rather than the inactive mutants of USP3, H56 A and C168 S could facilitate the proliferation of HepG2 cells, however Nultin-3 disrupted the ability of USP3 in promoting proliferation. Conclusion:1. USP3 directly targeted to MDM2.2. USP3 stabilized MDM2 protein via cleaving the k48-type polyubiquitination chain on MDM2.3. USP3 promoted the proliferation of HepG2 cells via MDM2.