关键词: USP10, 神经胶质母细胞瘤, 肿瘤增殖细胞周期, DNMT1

Abstract: Objective In order to investigate the role of the deubiquitinating enzyme USP10 in the malignant progression of glioblastoma and elucidate its underlying mechanisms. Methods The GEPIA2 database was used to analyze the mRNA expression of USP10 in glioblastoma samples and its relationship with the prognosis of patients compared with normal tissue samples. The CRISPR-Cas9 plasmid was designed to knock out the expression of USP10 in glioma cells.Cellular proliferation was assessed through clonal formation assays, EdU incorporation experiments, and flow cytometry analysis. Coimmunoprecipitation （Co-IP） assay combined with mass spectrometry was used to identify USP10 interacting proteins. Two siRNAs were designed to knock down the expression of endogenous USP10 in glioma cells, and RNA-Seq analysis was used to compare the different RNA between before and after knockdown, and signaling pathway enrichment analysis was performed. Western blot （WB） analysis was conducted to evaluate protein expression levels in cells. Results Compared with adjacent tissues （n=207）, USP10 was significantly higher expressed in glioma cells （n=163, P<0.05）, and its high expression was significantly associated with poor prognosis. Knockout of USP10 led to a significant reduction in the proportion of glioma cells in the S phase （P<0.05）, decreased clonal formation （P<0.05）, and an even more pronounced decrease in colony formation under the same doses of ionizing radiation and TMZ （P<0.05）. RNAseq results indicated that USP10 affected multiple metabolic pathways related to cell proliferation. USP10 interacted with DNMT1 and was positively correlated with the expression level in glioma samples. WB analysis revealed a significant decrease in DNMT1 protein levels and a shortened half-life following USP10 knockdown; The growth kinetics of USP10 and DNMT1 proteins, as determined by the HU release assay, were found to be synchronized with those of Cyclin E1. Flow cytometry analysis showed that USP10 knockout increased the proportion of cells in the G1 phase （P<0.05）, decreased the proportion in the S phase （P<0.05）, and increased the proportion in the G2 phase （P<0.05）. Additionally, the number of colony formation decreased when USP10 and DNMT1 were knocked down in glioma cells, and the proportion of colony formation decreased higher with the increase of IR and TMZ doses. Conclusion The deubiquitinating enzyme USP10 is overexpressed in glioblastoma and is linked to an unfavorable prognosis. USP10 promotes the transition of glioblastoma from G1 to S phase by enhancing the stability of DNMT1, thereby promoting the proliferative capacity of glioblastoma cells.

Key words: USP10, glioblastoma, tumor proliferation, cell cycle, DNMT1