目的探究核仁蛋白14(nucleolar protein 14,NOP14)过表达对卵巢癌SKOV3细胞增殖与迁移的影响及机制。方法检测卵巢癌细胞系(SKOV3、A2780、HO-8910、OVCAR)中NOP14的表达水平。SKOV3细胞中转染pcDNA-NOP14质粒以构建NOP14过表达细胞系...目的探究核仁蛋白14(nucleolar protein 14,NOP14)过表达对卵巢癌SKOV3细胞增殖与迁移的影响及机制。方法检测卵巢癌细胞系(SKOV3、A2780、HO-8910、OVCAR)中NOP14的表达水平。SKOV3细胞中转染pcDNA-NOP14质粒以构建NOP14过表达细胞系。细胞克隆形成实验和5-乙炔基-2′-脱氧尿苷(5-Ethynyl-2′-deoxyuridine,EdU)染色检测细胞增殖能力。无血清成球培养分析肿瘤干细胞特性。流式细胞术检测CD133阳性细胞比例。细胞形态学观察及上皮-间质转化(epithelial-mesenchymal transition,EMT)标志蛋白(E-cadherin、N-cadherin、Vimentin)检测评估EMT进程。蛋白质印迹和RT-qPCR检测核受体相互作用蛋白1(nuclear receptor interacting protein 1,NRIP1)及Wnt、β-catenin的表达水平。结果在卵巢癌细胞系中NOP14 mRNA和蛋白表达均显著低于正常卵巢上皮细胞。NOP14过表达后抑制SKOV3细胞增殖、干细胞特性及EMT转化(间质标志物N-cadherin、Vimentin下调,上皮标志物E-cadherin上调),以及NRIP1、Wnt和β-catenin的表达水平。结论NOP14可负向调控NRIP1和Wnt及β-catenin的表达,并抑制卵巢癌SKOV3细胞的增殖、干细胞特性及EMT进程。展开更多
Post-translational modification of spastin enables precise spatiotemporal control of its microtubule severing activity.However,the detailed mechanism by which spastin turnover is regulated in the context of neurite ou...Post-translational modification of spastin enables precise spatiotemporal control of its microtubule severing activity.However,the detailed mechanism by which spastin turnover is regulated in the context of neurite outgrowth remains unknown.Here,we found that spastin interacted with ubiquitin and was significantly degraded by K48-mediated poly-ubiquitination.Cullin3 facilitated spastin degradation and ubiquitination.RING-box protein 1,but not RING-box protein 2,acted synergistically with Cullin3 protein to regulate spastin degradation.Overexpression of Culin3 or BRX1 markedly suppressed spastin expression,and inhibited spastin-mediated microtubule severing and promotion of neurite outgrowth.Moreover,USP14 interacted directly with spastin to mediate its deubiquitination.USP14 overexpression significantly increased spastin expression and suppressed its ubiquitination and degradation.Although co-expression of spastin and USP14 did not enhance microtubule severing,it did increase neurite length in hippocampal neurons.Taken together,these findings elucidate the intricate regulatory mechanisms of spastin turnover,highlighting the roles of the Cullin-3–Ring E3 ubiquitin ligase complex and USP14 in orchestrating its ubiquitination and degradation.The dynamic interplay between these factors governs spastin stability and function,ultimately influencing microtubule dynamics and neuronal morphology.These insights shed light on potential therapeutic targets for neurodegenerative disorders associated with spastin defects.展开更多
文摘目的探究核仁蛋白14(nucleolar protein 14,NOP14)过表达对卵巢癌SKOV3细胞增殖与迁移的影响及机制。方法检测卵巢癌细胞系(SKOV3、A2780、HO-8910、OVCAR)中NOP14的表达水平。SKOV3细胞中转染pcDNA-NOP14质粒以构建NOP14过表达细胞系。细胞克隆形成实验和5-乙炔基-2′-脱氧尿苷(5-Ethynyl-2′-deoxyuridine,EdU)染色检测细胞增殖能力。无血清成球培养分析肿瘤干细胞特性。流式细胞术检测CD133阳性细胞比例。细胞形态学观察及上皮-间质转化(epithelial-mesenchymal transition,EMT)标志蛋白(E-cadherin、N-cadherin、Vimentin)检测评估EMT进程。蛋白质印迹和RT-qPCR检测核受体相互作用蛋白1(nuclear receptor interacting protein 1,NRIP1)及Wnt、β-catenin的表达水平。结果在卵巢癌细胞系中NOP14 mRNA和蛋白表达均显著低于正常卵巢上皮细胞。NOP14过表达后抑制SKOV3细胞增殖、干细胞特性及EMT转化(间质标志物N-cadherin、Vimentin下调,上皮标志物E-cadherin上调),以及NRIP1、Wnt和β-catenin的表达水平。结论NOP14可负向调控NRIP1和Wnt及β-catenin的表达,并抑制卵巢癌SKOV3细胞的增殖、干细胞特性及EMT进程。
基金supported by the National Natural Science Foundation of China,No.32071033(to MT)Basic and Applied Basic Research Foundation of Guangdong Province,Nos.2023A1515010140(to MT),2022A1515140169(to MT),2022A1515111096(to ZC)+3 种基金Science and Technology Project of Guangzhou,Nos.202201010015(to YL),2023A03J0790(to TJ)Basic and Applied Basic Research Foundation of Guangzhou,No.2023A04J1285(to ZC)Medical Research Foundation of Guangdong Province,No.A2023147(to ZC)Health Science and Technology Project of Guangzhou,No.20221A011039(to TJ)。
文摘Post-translational modification of spastin enables precise spatiotemporal control of its microtubule severing activity.However,the detailed mechanism by which spastin turnover is regulated in the context of neurite outgrowth remains unknown.Here,we found that spastin interacted with ubiquitin and was significantly degraded by K48-mediated poly-ubiquitination.Cullin3 facilitated spastin degradation and ubiquitination.RING-box protein 1,but not RING-box protein 2,acted synergistically with Cullin3 protein to regulate spastin degradation.Overexpression of Culin3 or BRX1 markedly suppressed spastin expression,and inhibited spastin-mediated microtubule severing and promotion of neurite outgrowth.Moreover,USP14 interacted directly with spastin to mediate its deubiquitination.USP14 overexpression significantly increased spastin expression and suppressed its ubiquitination and degradation.Although co-expression of spastin and USP14 did not enhance microtubule severing,it did increase neurite length in hippocampal neurons.Taken together,these findings elucidate the intricate regulatory mechanisms of spastin turnover,highlighting the roles of the Cullin-3–Ring E3 ubiquitin ligase complex and USP14 in orchestrating its ubiquitination and degradation.The dynamic interplay between these factors governs spastin stability and function,ultimately influencing microtubule dynamics and neuronal morphology.These insights shed light on potential therapeutic targets for neurodegenerative disorders associated with spastin defects.
