Functional Analysis Of Deubiquitinase USP7 In Acute T-cell Lymphoblastic Leukemia

Background and Objective:Acute T-cell lymphoblastic leukemia is an immature lymphoma caused by the malignant diffusion of T progenitor cells into the bone marrow.The prognosis of different T-ALL patients varies greatly,and the situation of patients with T-ALL resistance or relapse is not optimistic.The current research is mainly to find out more effective and less toxic anti-leukemia targeted drugs,which requires higher specificity of the drug,so it also requires a more accurate understanding of the pathogenesis of T-ALL.In most cases of T-ALL,NOTCH1 is activated by mutation.Previous researchers have discovered the main carcinogenic mechanism of NOTCH1 in leukemia in acute T-cell lymphoblastic leukemia.Our work sought to find NOTCH1 cofactor proteins with functions of cancer-promoting that can be targeted to treat T-ALL without toxicity.We have discovered a new member of T-ALL that affects the amplification of NOTCH1-positive T-ALL cells.A deubiquitinase(DUB)protein that plays a key role in leukemia,known as ubiquitin-specific protease 7(USP7).The purpose of this study is to investigate the effects of USP7 on T-cell acute lymphoblastic leukemia cells,and to explore the molecular mechanisms of this effect in order to find new targets,and to develop more targeted,less side-effect targeted therapeutic drugs for the treatment of acute T-cell lymphocytic leukemia.Methods:Effects on the growth of T-ALL cells in which USP7 and USP13 have been silenced was detected by cell counting and apoptosis detection technology;The binding between USP7 and ICN1/MYB was detected by co-immunoprecipitation;the effect of silencing USP7 on ICN1/MYB protein expression was detected by Western blot and RNA interference;the efficiency of silencing USP7 was detected by Real-time PCR;ICN1 protein antibodies prepared by extensive purification of E.coli expression plasmids and polyclonal antibody technology.Results:We verified that knocking down USP7 slows the growth of T-ALL cells.Thenwe demonstrated the direct binding of ICN1 and USP7 by immunoprecipitation experiments,and this conclusion led us to explore the molecular mechanism and biological function of this effect.Finally,we verified through a series of experiments that USP7 can regulate the protein expression of MYB.Therefore,we can control the NOTCH1 pathway and ultimately inhibit the oncogene transcriptional pathway in T cell acute lymphoblastic leukemia.Finally,inhibition of the USP7 may be an effective therapeutic target for this disease.Western blot showed that the rabbit antibody immunized with ICN1 antigen peptide protein could bind to ICN1 extracted from ICN1 highly expressed T-ALL cell in vitro,and confirmed that the protein expressed by expression plasmid was the target protein and the activity of antibody obtained is correct.Conclusions:The deubiquitinated protein USP7 can directly bind to the oncoprotein NOTCH1 and regulate the expression of MYB.This regulation can directly affect the growth of T-ALL cells with high expression of NOTCH1.Based on the important regulation of NOTCH1 in T cell acute lymphoblastic leukemia,our findings provide a potential therapeutic target for the clinical treatment of T cell acute lymphoblastic leukemia with high expression of NOTCH1.