Ubiquitin-specific Protease USP38 Regulates Proliferation And Metastasis Of Glioblastoma

Glioblastoma(GBM),the most malignant glioma,is characterized by a high degree of proliferation and invasion of surrounding normal tissue to form blood vessels.The annual incidence of glioblastoma is about 1/20,000,with more than 14,000 new cases annually,which is common in the olderly.Glioblastoma cells have strong migration and invasion ability,and the lesions are located in the brain,so the prognosis is poor.The current survival rate of grade IV gliomas is about 13 months,and the clinical treatment is very difficult.Therefore,further identification of useful biomarkers is critical for the treatment of GBM.USP38 is a member of the USPs family that includes cysteine-type endopeptidase activity and thiol-dependent ubiquitinyl hydrolase activity.Previously,USP38 has been reported to be associated with the occurrence of primary breast cancer;In addition,USP38 is upregulated in the lung tissue of copd.Recently,it has been reported that USP38 is a LSD1-specific deubiquitinase,which affects cell physiology by interacting with LSD1.Although USP38 has been reported in primary breast cancer,chronic obstructive pulmonary disease and other diseases,its role in the development of glioblastoma has hardly been reported.In this study,we analyzed R2 database and found that USP38 was highly expressed,and patients with GBM had poor prognosis and short survival.Meanwhile,we detected the expression levels of USP38 in different glioblastoma cell lines and astrocytes.Next,we knocked down and overexpressed USP38 to detect its influence on cell proliferation,apoptosis,migration and invasion,and tumorigenesis ability in vivo and in vitro,so as to provide new ideas for the treatment of glioblastoma.Our experimental results are as follows: 1.The expression of USP38 in glioblastoma is closely related to the prognosis of patientsAccording to the analysis of R2 data platform,the expression level of USP38 in glioblastoma with a higher degree of malignancy was higher.It was detected by qrt-pcr and Western blot that USP38 was generally expressed in glioblastoma,but not in normal glial cells,and the expression level was closely related to the degree of malignancy of the tumor.2.USP38 promotes the proliferation and tumorigenesis of glioblastomaTo explore the role of USP38 in the proliferation and tumorigenesis of glioblastoma cells,We knocked down USP38 using lentiviral-mediated shRNA interference.Western Blot showed that after we knocked down USP38 successfully,morphological observation of cells showed that the growth of glioblastoma cells was significantly inhibited.Then we used the BrdU incorporation assay to detect cellular DNA synthesis.The results showed that the DNA synthesis of the USP38 interference group was significantly inhibited.Next,we used flow cytometry and Western Blot to detect the cell cycle and related cyclins,respectively.It was found that after USP38 interference,cell cycle arrest was in G0/G1 phase,and related cyclins such as CDK2,CDK4,etc.have undergone significant downward adjustments.Then we used soft agar cloning experiments,mouse subcutaneous tumor formation experiments and immunohistochemistry(IHC)experiments to detect the tumorigenic ability of glioblastoma cells in vitro and in vivo,and found that when we knocked down USP38,the gelatinous The ability of tumor cells to form tumors in vitro and in vivo is reduced.Although we designed two interference fragments,since shRNA is prone to off-target effects,we performed a full-length USP38 recovery on cell lines that knock down USP38,ensuring that changes in physiological functions are caused by knocking down USP38,and detection.Whether the relevant physiological functions of the cells were restored after USP38 was restored.Experiments confirmed that after USP38 was restored,the relevant physiological functions of the cells were restored.The above experiments indicate that the ubiquitin-specific protease USP38 can promote the proliferation and tumor formation of glioblastoma cells.3.USP38 promotes the migration and invasion of glioblastoma cellsThe extremely strong metastatic ability of tumor cells is a very important reason for its difficulty in curing.In order to examine the effect of USP38 on the metastatic ability of glioblastoma cells,we used different cell lines that knocked down USP38 and and then restore the USP38 for migration and invasion assays.The results showed that the migration and invasion ability of the cells were inhibited after interference with USP38.Moreover,the expression of mesenchymal markers Snail,Slug and other proteins were inhibited after interference with USP38.When USP38 was restored,the migration and invasion ability of glioblastoma was restored.4.USP38 inhibits apoptosis in glioblastoma cellsTo investigate the effect of knockdown of USP38 on apoptosis in glioblastoma cell lines,we performed flow cytometry and Western Blot experiments on LN-229 and U-87 MG cell lines that interfered with USP38.The results showed that after interfering with USP38,the expression of important anti-apoptotic factor Bcl2 in the anti-apoptotic family of cells decreased significantly,and the cells showed obvious apoptosis;while the mitochondrial apoptosis pathway-related protein C-caspase3,CCaspase9 and C-PARP were significantly up-regulated,indicating that apoptosis may be through the mitochondrial apoptotic pathway.Apoptosis of glioblastoma was restored when USP38 was restored.5.USP38 regulates the expression of the proto-oncogene c-Myc in glioblastomaThe proto-oncogene c-Myc was confirmed to be a proto-oncogene in glioblastoma.After USP38 interference,we found that m-RNA levels and protein levels of c-Myc were significantly down-regulated by qRT-PCR and Western Blot experiments.When we looked at GeneCards,we predicted that c-Myc might be combined with the promoter region of USP38,and we looked at the literature on USP38 and found that its function was essentially at the protein level.So on the question of how to down-regulate the expression of c-Myc after knocking down USP38,we first did not consider this issue from the transcription level,but considered it from the post-transcriptional level.Next we did an IP experiment to see if there is any interaction between USP38 and c-Myc.The experimental results show that USP38 and c-Myc are bound and interact.However,how USP38 regulates the balance between ubiquitination and deubiquitination of c-Myc requires further study of its mechanism to explain.In summary,USP38 may affect the expression of c-Myc at the post-transcriptional level by affecting the ubiquitination of c-Myc,thereby affecting the proliferation and migration of glioblastoma.This study provides an important basis for USP38 as a marker for glioblastoma and enriches the theoretical basis for molecular therapy of glioblastoma.