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【Objective】 To explore the expression of USP9X in platelets and its effect on platelet function. 【Methods】 The expression of USP9X in human and mouse was evaluated by PCR and Western blot. Platelets from young and old mice were separated and prepared, and the expression of USP9X was detected. USP9X inhibitos were used to assess the regulation of USP9X in platelet function, including aggregation, ATP release and spreading. Platelet lysates were collected in different time points to evaluate the change of phosphorylation of Akt in USP9X inhibitors treated platelets. 【Results】 Both human and mouse platelets expressed USP9X. Compared to the young mice, the old mice showed significantly enhanced expression of USP9X(P<0.05). To assess the effect of USP9X on platelet function, USP9X inhibitor was used to pre-incubate platelets for 30 min and platelet function were examined later. Results showed that USP9X inhibitor significantly decreased platelet activation including aggregation, ATP release and spreading(P<0.05). Compared to the control group, the inhibitor treated group showed a significant decrease in the spreading area after 45 minutes. The Western blot results showed a significant decrease in Akt phosphorylation levels of platelets in the USP9X inhibitor treated group. 【Conclusion】 Both human and mouse platelet express USP9X, and inhibition of USP9X decreased platelet function including aggregation, ATP release and spreading. USP9X can also influence the phosphorylation of Akt. The inhibitor of USP9X may become a potential therapeutic target for thrombosis intervention.
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Objective:To explore whether the deubiquitinating enzyme deubiquitin ligase 9X(USP9X)affects the proliferation of nasopharyngeal carcinoma(NPC)cells via regulating forkhead box Ml(FOXM1)mediated glycolysis. Methods:Western blotting and real-time fluorescence quantitative PCR,respectively,were used to detect the protein and mRNA expression levels of USP9X in NPC tissue and normal nasal mucosa tissue.NPC cells(CNE2 and HNE2)were infected with lentivirus carrying specific shRNA targeting USP9X gene(shUSP9X)or full-length USP9X gene(Flag-USP9X)for USP9X overexpression.CCK-8 assay was used to evaluate the effect of USP9X silencing or overexpression on the proliferation of NPC cells.Cellular energy metabolism assay and extracellular acidification rate(ECAR)assay were performed to investigate the impact of USP9X alteration on the glycolysis of NPC cells.The interaction between USP9X and FOXM1 was analyzed using ubibrowser_v3/database,co-immunoprecipitation,and ZDOCK software,and the changes in FOXM1 protein ubiquitination levels upon USP9X silencing was examined.Real-time fluorescence quantitative PCR and Western blotting,respectively,were used to detect the effect of USP9X alteration on the expression of FOXM1 mRNA and protein in NPC cells.Finally,FOXM1 expression was restored in USP9X silencing CNE2 cells by expression of recombinant plasmid Flag-FOXM1 carrying full-length FOXM1 gene through lentiviral infection.CCK-8 assay was used to evaluate the effect of FOXM1 upregulation on the proliferation of CNE2 cells.Cellular energy metabolism assay and ECAR assay were used to investigate the effect of FOXM1 upregulation on the glycolysis of CNE2 cells. Results:Compared with normal nasal mucosa tissue,the expression levels of USP9X mRNA and protein were significantly increased in NPC tissues(P＜0.05).After USP9X downregulation,the proliferation and glycolysis of CNE2 cells was significantly inhibited as indicated by the results of CCK-8 assay,cellular energy metabolism assay and ECAR assay(P＜0.05).In contrast,the proliferation and glycolysis of HNE2 cells was significantly enhanced after USP9X upregulation(P＜0.05).The interaction between USP9X and FOXM1 was confirmed by ubibrowser_v3/database,co-immunoprecipitation,and ZDOCK software analysis.Silencing USP9X could significantly increase FOXM1 ubiquitination in CNE2 cells.Overexpressing FOXM1 in low-USP9X CNE2 cells restored the proliferation and glycolysis activity of NPC cells(P＜0.05). Conclusion:USP9X can enhance NPC cell glycolysis by inhibiting FOXM1 ubiquitination and subsequent degradation,thereby promoting NCP cell proliferation.USP9X may be a potential novel therapeutic target for the treatment of NPC.
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Objective:To summarize and analyze the clinical and genotype features of female-restricted X-linked syndromic mental retardation-99(MRXS99F, OMIM: 300968)caused by USP9 X gene mutation, and to improve the clinicians′ understanding of the disease. Methods:Clinical data and genotypes of 2 children with MRXS99F treated in the Children′s Hospital of Nanjing Medical University in March 2020 (case 1) and June 2020 (case 2) were analyzed, and the relevant databases at home and abroad were reviewed to summarize the clinical characteristics and gene variation characteristics of the disease.Results:The 2 cases were 6 months old (case 1) and 5 years old (case 2), both showed psychomotor retardation.Case 1 presented a short stature, pigment abnormality, characteristic facial features, hypotonia, recurrent respiratory tract infections, laryngeal cartilage hypoplasia, atrial septal defect, feeding difficulty, hearing loss and brain hypoplasia.Case 2 had abnormal electroencephalogram.As confirmed by whole-exome sequencing, two children carried c. 6972+ 1G>A, c.6437C>T of USP9 X, respectively.Neither of the 2 variations was previously reported.Twenty-two cases of MRXS99F caused by USP9 X gene mutation were reported in 4 literatures globally, and 24 cases were combined with this study.The clinical manifestations of 20/22 children had special faces.All of them accompanied mental retardation combined with motor and language retardation, and carried neonatal variation. Conclusions:This is the first case report of MRXS99F induced by USP9 X gene variation in China.MRXS99F caused by functional deletion and variation of USP9 X gene is mainly characterized by psychomotor retardation, language disorder, special face and multiple congenital malformations.For children with unexplained growth retardation, special face and multiple congenital malformations, genetic testing like high-throughput sequencing should be carried out as early as possible to determine the etiology.