去泛素连接酶USP9X通过调控FOXM1介导糖酵解影响鼻咽癌细胞增殖的机制研究

The regulatory mechanism of deubiquitin ligase USP9X in nasopharyngeal carcinoma cell proliferation through FOXM1-mediated glycolysis

目的:探讨去泛素连接酶USP9X(deubiquitin ligase 9X)是否通过调控叉头框蛋白M1(forkhead box M1,FOXM1)介导糖酵解从而影响鼻咽癌细胞的增殖能力。方法:采用实时荧光定量PCR法和蛋白质印迹法检测鼻咽癌组织和正常鼻黏膜组织以及鼻咽癌细胞中USP9X mRNA和蛋白的表达情况。通过病毒感染的方法将携带有特异性针对USP 9X基因的shRNA(shUSP9X)和携带有USP9X全基因的重组质粒Flag-USP9X(过表达USP9X)分别转入鼻咽癌CNE2和HNE2细胞,采用CCK-8法检测沉默或过表达USP 9X基因对鼻咽癌细胞增殖的影响,通过对细胞能量代谢和细胞外酸化率(extracellular acidification rate,ECAR)的检测分析对鼻咽癌细胞糖酵解能力的影响。通过ubibrowser_v3/数据库、免疫共沉淀和ZDOCK软件分析USP9X与FOXM1蛋白的结合情况,并通过泛素化实验观察沉默USP9X对FOXM1蛋白泛素化水平的影响;随后进一步通过实时荧光定量PCR法和蛋白质印迹法检测对FOXM1 mRNA和蛋白表达的影响。最后,采用慢病毒感染的方法在沉默USP9X表达的CNE2细胞中同时转入重组载体Flag-FOXM1恢复FOXM1的表达,再通过CCK-8法、细胞能量代谢和ECAR检测分析上调FOXM1对CNE2细胞增殖和糖酵解能力的影响。结果:与正常鼻黏膜组织相比,鼻咽癌组织中USP9X mRNA和蛋白的表达水平均显著提高(P均<0.05)。下调鼻咽癌CNE2细胞中USP9X的表达水平后,CCK-8法、细胞能量代谢检测和ECAR实验检测发现,CNE2细胞的增殖和糖酵解能力均被明显抑制(P均<0.05)。相反,上调HNE2细胞中USP9X的表达水平后,HNE2细胞的增殖和糖酵解能力均被明显提高(P均<0.05)。通过ubibrowser_v3/数据库、免疫共沉淀和ZDOCK软件证实,USP9X与FOXM1蛋白存在相互结合的关系,CNE2细胞中沉默USP9X表达水平可显著增加FOXM1的泛素化水平。在低表达USP9X的CNE2细胞中过表达FOXM1可恢复鼻咽癌细胞的增殖和糖酵解能力(P均<0.05)。结论:去泛素连接酶USP9X通过抑制FOXM1泛素化降解进而提高鼻咽癌细胞的糖酵解能力,最终促进鼻咽癌细胞的增殖。USP9X可能是鼻咽癌治疗上一个潜在的新靶点。

Objective:To explore whether the deubiquitinating enzyme deubiquitin ligase 9X(USP9X)affects the proliferation of nasopharyngeal carcinoma(NPC)cells via regulating forkhead box M1(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 USP 9X gene(shUSP9X)or full-length USP 9X 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 FOXM 1 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.