Overexpression Of Ubiquitin Specific Proteases 44 Promotes The Malignancy Of Glioma By Stabilizing Tumor-promoter Securin

Glioblastoma is one of the most common primary tumors of the central nervous system.The effective therapeutic method has yet not been found in traditional therapeutics of GBM due to its infiltrative growth,ill-defined margin with the surrounding brain tissue,and the tolerance of radiotherapy and chemotherapy.The current individualized genomic therapy is expected to be one of the ways to cure glioblastoma,but there is still lack of an ideal and reliable therapeutic target.Therefore,it has become one of the hot spots in the field of brain tumor research to find the molecular target for regulating the malignant biological behavior of glioblastoma and to clarify its mechanism in the pathogenesis of GBM.In the cell cycle process,timely and accurate separation of sister chromatid is critical to the stability of cell genetics.The spindle assembly checkpoint(SAC)is an important mechanism to monitor ensure the proceeding of mitosis,the abnormality of SAC is an important reason leading to the formation of aneuploid cells and even tumor.The previous research reported that the important regulatory proteins of the spindle assembly checkpoint——deubiquitinase USP44 was closely associated with tumor.Deubiquitinase USP44 can stable the protein level of securin until all kinetochores matched spindle correctly in normal cell cycle progression,which prevented immature mitosis.Inhibition of the expression of USP44 in mouse embryonic fibroblasts could significantly increase the proportion of aneuploid cells and the instability of chromosomes,making it more prone to malignant transformation.USP44 is the antagonist of tumor promoting factor UbcH10,but USP44 is up-regulated in tumor cells.Due to the lack of effective specific antibody of endogenous USP44,whether the overexpression of USP44 is involved in maintaining the malignant proliferation of tumor cells and the associated regulation mechanism is unclear.Our experiment start from screening and identifying the specific antibody of endogenous USP44 to the analyze the correlation between the USP44 expression level and the pathologic grade of glioma tissues,then analyze the effect of USP44 on the malignant biological behavior of GBM and carry out part of the signal pathways that USP44 might be involved in.Part ? Screening and identification of USP44 specific antibodiesObjective: screen and identify specific antibodies to recognize endogenous USP44 effectively by molecular biological techniques.Methods: prepared three kinds of antibodies: Abnova pAb21808,Santa Cruz sc-337203 and Origene TA801913.Detected whether the three antibodies can recognize endogenous USP44 specifically in U251 MG,U87MG and A172 cells by western blot;overexpress Flag-tagged USP44 fusion protein in 239 T cells,fluorescence signals of USP44 and Flag were detected by immunofluorescence assay;in U251 MG cells,detect the fluorescence signals of USP44 and B23 by immunofluorescence with Alexa Fluor 488 and Alexa Fluor 594 labeled secondary antibodies respectively;detected the efficiency of USP44 knockdown by western blot and immunofluorescence assay with the three antibodies..Results: in immunoblotting experiments,the three kinds of antibodies can specifically recognize endogenous USP44 in three cell lines,formatting the only blot bands;the fluorescence signals of pAb21808 and anti-Flag antibody were completely overlapped;fluorescence signals of pAb21808 antibody and anti-B23 antibody were coincident in the nucleolus;in western blot assay,all the three kinds of antibodies can detect decreased expression of USP44 caused by shRNA.Knockdown of USP44 significantly decreased the fluorescence signal in immunofluorescence assay with the antibody pAb21808.Conclusions: antibody pAb21808 has the best specificity to recognize the endogenous USP44 by detecting the subcellular localization of USP44 and analyzing the efficiency of siRNA accurately,therefore we chose antibody pAb21808 to perform the following experiments.Part ? The relationship between the expression level of USP44 and the pathological grade,prognosis of gliomaObjective: evaluated the relationship between the expression level of USP44 and the pathological grade and prognosis of glioma tissue samples from the protein and mRNA levels.Methods: the expression level of USP44 protein in tissue microarray containing 61 samples of different grade gliomas were assayed by immunohistochemical staining with pAb21808 antibody;the expression level of USP44 mRNA were detected in 40 cases of different grade frozen glioma tissues by qRT-PCR assay;survival analysis was performed with the data of qRT-PCR.Results: the expression of USP44 protein was correlated with the pathological grades of glioma;the expression level of USP44 mRNA in high-grade gliomas was significantly higher than that in low-grade gliomas;the patients with high USP44 mRNA expression suffered the significantly shorter overall survival time than patients in low expression group.Conclusions: the expression level of USP44 increased with increasing of the pathological grade of glioma,and the high expression of USP44 suggested a poor prognosis.Part ? Effects of USP44 on the malignant biological behavior of GBM cellsObjective: analyze the changes of proliferation,invasion,cell cycle and apoptosis in GBM cells caused by knockdown of USP44.Methods: U251 MG and A172 cells were transfected with USP44-shRNA lentivirus;WST-1 assay and colony formation assay were performed to detect proliferation in the transfected cells;changes in migration and invasion were detected by transwell assay;stained the transfected U251 MG and A172 cells with propidium iodide(PI)and analyzed cell cycle distribution by FCM;analyzed percentage of apoptosis in transfected cells stained with PE/7-AAD by flow cytometry;detected the cycle and apoptosis associated protein by immunoblotting with specific antibodies.Results: knockdown of USP44 significantly inhibit the proliferation,migration and invasion in U251 MG and A172 cells,arrested cell cycle in G2/M phase and increased the proportion of apoptotic cells.Conclusions: knockdown of USP44 can significantly inhibit the malignant biological behavior of GBM cells,block cell cycle in G2/M phase and induce apoptosis.Part ? Interaction between USP44 and tumor promoting factor securinObjective: Verify whether there is an interaction between USP44 and securin,and whether USP44 can stabilize securin by deubiquitination.Methods: detect the fluorescence signal localization of USP44 and securin in U251 MG cells by immunofluorescence;confirmed the direct combination of USP44 and securin by immunoprecipitation assay;treated USP44-shRNA-transfected U251 MG cells with proteasome inhibitor MG132 in different concentration,the expression of securin was detected by western blot assay.Results: the fluorescence signals of USP44 and securin overlapped obviously in mitotic cells;IP assay confirmed that endogenous USP44 can directly combine with securin;MG132 can reverse the down-regulation of securin caused by knockdown of USP44 without affecting the expression level of USP44 itself.Conclusions: Securin is a substrate of USP44,and USP44 can stabilize securin and inhibit its degradation by deubiquination.Part ? Effect of knockdown USP44 on tumorigenicity of U87MG cells in vivoObjective: Investigate the effect of down-regulating USP44 expression on the tumorigenicity of GBM cells in vivo.Methods: transfected U87 MG cells with lentivirus of USP44-shRNA and cultured continuously;subcutaneously injected the single-cell suspension(107cells)of USP44-shRNA-U87 MG and USP44-NC-U87 MG cells on both sides of the proximal hindlimb in 5-week-old male nude mice respectively;measured the major axis and minor axis of tumor with caliper every 2 days.When the major axis of tumor was longer than 10 mm,the nude mice were killed off,and the tumors were removed to made paraffin sections.Immunohistochemistry assay was performed to detected the cell morphology and the expression level of Ki-67 in the sections.Results: the final volume and growth rate of the tumor were significantly reduced in the USP44-knockdown group compared with the control group.The results of immunohistochemistry indicated that the expression level of Ki-67 in USP44-knockdown group was significantly lower than that in the control group.Conclusions: knockdown of USP44 significantly inhibit the tumorigenicity of U87 MG cells in vivo.