The Role And Molecular Mechanism Of Ubiquitin Specific Peptidase 10 Enhancing The Stability Of CCND1

ObjectivesCyclin D1 is an important regulatory point to ensure accurate DNA replication and cell mitosis.Under the stimulation of growth factors,CCND1 assembles with the cyclin kinase CDK4/6 to form a kinase complex,which phosphorylates the retinoblastoma tumor suppressor protein Rb,freeing the transcription factor E2F,so that the cell cycle moves from G1 to S.Under normal physiological conditions,after the cell cycle enters the S phase,CCND1 is degraded by the ubiquitin proteasome pathway,but the degradation pathway of CCND1 is destroyed in tumor tissues,and a large amount of CCND1 protein is accumulated,making cell proliferation uncontrolled.Therefore,the ubiquitin-proteasome pathway of CCND 1 plays a key role in tumorigenesis and development.However,less research has been done on deubiquitinating enzymes that increase the stability of CCND1.This study aims to discover the deubiquitinating enzyme of CCND 1 and explore its mechanism of action,providing a new direction for the treatment of glioma cells.Methods1.To verify the interaction of USP10 and CCND1 proteins in HEK293T cells,U251 cells and HS683 cells by Co-Immunoprecipitation(Co-IP)method;2.After overexpressing or knocking down USP10,Western Blot method was used to explore the effect of CCND1 protein expression from both endogenous and exogenous aspects;3.Co-Immunoprecipitation(Co-IP)technique was used to detect the effect of USP 10 on the ubiquitination of exogenous CCND1 protein and the type of ubiquitination.The USP 10 cDNA fragment was inserted into pLVX-AcGFP lentiviral vector and viruses were obtained 72 hrs later from transfected cells and were applied to infect the appropriate cells.4.The stability and half-life of CCND1 protein were analyzed with CHX chase experiment and western blotting methods in the presence of USP 10;5.Using CRISP Cas9 technology,USP 10 lentivirus was constructed to infect glioma cells,followed by WB technology and flow cytometry to analyze the apoptosis of glioma cells in the absence of USP10;6.Using MTT colorimetry and WB technology to screen USP10 inhibitors from the FDA-approved molecule drug library.U251 and HS683 cells were treated with acevaltrate and the expression of CCND1 and USP10 proteins was detected by immunoprecipitation and WB.The ubiquitination level of CCND1 was detected by immunoprecipitation and WB;7.U251 and HS683 cells were treated with acevaltrate and RT-PCR assay was performed to analyze the mRNA level of USP10 and CCND1;8.Flow cytometry and MTT assay were performed to analyze the apoptosis of glioma cells induced by acevaltrate.Results1.USP 10 and CCND1 were overexpressed in HEK293T,U251 and HS683cells,and co-immunoprecipitation experiments showed that USP 10 and CCND1 could bind to each other.2.After overexpression of USP 10 and CCND1 in HEK293T,U251 and HS683 cells,the IP/WB results showed that USP 10 reduced the polyubiquitination of CCND1 and increase the stability of protein CCND 1.At the same time,further experiments showed that the ubiquitination site of USP 10 was mainly K48.3.In the context of the overexpression of USP10 and CCND1 in HEK293T cells,USP10 could significantly increase the protein level of CCND1 in a concentration-dependent manner.Experiments show that USP 10 inhibits the degradation of CCND1 protein through the ubiquitin proteasome pathway and increases protein expression4.CHX experimental results show that USP 10 can increase the protein level of CCND1 and extend the half-life of CCND1.5.After knocking down USP10 in U251 cells and HS683 cells,the results showed that knockdown of USP 10 can reduce the protein level of CCND1,and flow cytometry results showed that knockdown of USP 10 can induce apoptosis of glioma cells.6.MTT assay and WB analyses showed that only acevaltrate(AVT)promoted USP 10 CCND1 protein degradation.At the same time,experimental results show that AVT can increase the ubiquitin of CCND1 protein.But the increasing ubiquitination level can be reversed by USP 10.7.The results of RT-PCR experiments showed that AVT did not affect the mRNA expression levels of USP10 and CCND18.Flow cytometry and MTT experiments showed that AVT can induce the apoptosis of U251 cells and HS683 cells and exert anti-glioma activity.Conclusion1.USP10 can interact with CCND1 to form a complex in vivo and in vitro2.USP10/CCND1 complex significantly inhibits the polyubiquitination of CCND1 and increase the stability of protein CCND1.Knockdown of USP10 in U251 cells and HS683 cells can reduce endogenous CCND1 protein levels and induce apoptosis in glioma cells.The results will help to provide new strategies for clinical treatment of gliomas.