USP11调控VEGF表达及血管形成促进肝癌细胞的侵袭和转移

目的:探究去泛素化酶11(USP11)调控VEGF和血管形成促进肝癌细胞侵袭和转移能力的机制。方法:运用Real-time PCR实验检测USP11对肝癌细胞中VEGF基因表达水平的影响;应用Elisa实验检测USP11对肝癌细胞分泌VEGF蛋白的影响;利用对照组和USP11敲低组肝癌细胞分泌上清分别处理血管内皮细胞HUVECs,运用Transwell基质胶实验检测血管内皮细胞的迁移能力;运用CCK-8实验检测血管内皮细胞的增殖能力;运用小管形成实验检测血管内皮细胞的成管能力。结果:敲低USP11可降低肝癌细胞中VEGF的基因表达水平(P<0.01),并抑制肝癌细胞分泌VEGF蛋白(P<0.001);与对照组相比,敲低USP11的肝癌细胞分泌上清处理血管内皮细胞,血管内皮细胞的增殖能力减弱(P<0.01),成管能力也远低于对照组;两组肝癌细胞和血管内皮细胞共培养,基质胶实验表明,敲低USP11组,血管内皮细胞的迁移能力降低(P<0.01)。结论:USP11可以增加VEGF的基因转录和蛋白表达水平,增强血管内皮细胞的增殖、迁移和成管能力,进而促进肝癌细胞的侵袭和转移。

rAAV9介导的siRNA靶向USP11调控2型糖尿病大鼠血糖及视网膜NF-κB表达的机制研究

目的 探讨9型重组腺相关病毒介导的去泛素化酶siRNA基因敲除对2型糖尿病大鼠血糖及视网膜核因子κB表达的影响。方法 将48只大鼠按照随机数字表法分为对照组、2型糖尿病组、9型重组腺相关病毒组和9型重组腺相关病毒+去泛素化酶敲低组。2型糖尿病组、9型重组腺相关病毒组和9型重组腺相关病毒+去泛素化酶敲低组大鼠采用链脲佐菌素腹腔注射诱导2型糖尿病大鼠模型,对照组大鼠仅注射等剂量生理盐水,造模成功后尾静脉分别注射生理盐水、9型重组腺相关病毒慢病毒载体和9型重组腺相关病毒+去泛素化酶敲低慢病毒载体。造模4周及8周末检测各组大鼠体质量和血糖、血脂水平,收集视网膜切片,采用免疫组织化学法和免疫印记法检测核因子κB的表达。结果 造模前后4组大鼠体质量比较差异有统计学意义(P<0.01),造模4周及8周末4组间比较差异有统计学意义(P<0.01),对照组最高,2型糖尿病组最低。造模前后4组大鼠血糖水平比较差异有统计学意义(P<0.05或0.01),造模4周及8周末4组间比较差异有统计学意义(P<0.01),2型糖尿病组最高,对照组最低。造模前后4组大鼠血清甘油三酯水平比较差异有统计学意义(P<0.05或0.01),造模4周及8周末4组间比较差异有统计学意义(P<0.01),2型糖尿病组最高,对照组最低。2型糖尿病组大鼠视网膜核因子κB阳性水平最高,其次为9型重组腺相关病毒组、9型重组腺相关病毒+去泛素化酶敲低组,对照组阳性水平最低。2型糖尿病组大鼠视网膜核因子κB蛋白表达最高,其次为9型重组腺相关病毒组、9型重组腺相关病毒+去泛素化酶敲低组,对照组最低。结论 9型重组腺相关病毒介导的去泛素化酶siRNA基因敲除可显著降低2型糖尿病大鼠血糖、血脂水平,并抑制视网膜核因子κB的表达,发挥保护视网膜的作用。

USP11调节IGF2BP3介导口腔鳞状细胞癌细胞增殖和侵袭机制研究

目的:探究去泛素化酶(USP11)通过调节胰岛素样生长因子2-mRNA结合蛋白3(IGF2BP3)表达影响口腔鳞状细胞癌(OSCC)恶性表型的分子机制。方法:免疫组化和Western blot检测OSCC患者(n=50)癌组织和癌旁组织中USP11蛋白表达,Western blot检测USP11蛋白在正常人口腔上皮角质细胞(HOK)和人OSCC细胞SCC-25和CAL-27中表达;Kaplan-Meier生存模型分析USP11高低表达对OSCC患者生存率的影响;siRNA USP11(si-USP11)转染SCC-25和CAL-27细胞敲低内源性USP11表达;CCK-8、划痕实验和Transwell实验检测细胞增殖、迁移和侵袭;Western blot检测敲低USP11后的IGF2BP3蛋白表达;敲低USP11和过表达IGF2BP3,检测OSCC细胞增殖、迁移和侵袭的变化;过表达USP11同时敲低IGF2BP3,检测敲低IGF2BP3对USP11过表达OSCC细胞增殖、迁移和侵袭的影响;裸鼠接种SCC-25细胞构建皮下移植瘤模型,考察敲低USP11对SCC-25细胞成瘤抑制作用。结果:与癌旁组织相比,OSCC患者癌组织中USP11蛋白免疫组化评分显著升高(P<0.01);与HOK细胞相比,SCC-25和CAL-27细胞USP11蛋白表达显著增加(P<0.01)。敲低USP11降低OSCC细胞增殖、迁移和侵袭能力(P<0.01),下调IGF2BP3蛋白表达。过表达IGF2BP3逆转USP11敲低对OSCC细胞增殖、迁移和侵袭能的抑制作用;敲低IGF2BP3逆转USP11过表达对OSCC细胞增殖、迁移和侵袭的促进作用。裸鼠致瘤性实验显示敲低USP11抑制移植瘤模型中SCC-25细胞体内生长。结论:USP11在OSCC患者癌组织中表达升高且高表达患者预后更差,USP11通过上调IGF2BP3蛋白表达促进OSCC细胞增殖、迁移和侵袭。

A non-metabolic function of hexokinase 2 in small cell lung cancer:promotes cancer cell stemness by increasing USP11-mediated CD133 stability

Background:Maintenance of cancer stem-like cell(CSC)stemness supported by aberrantly regulated cancer cell metabolism is critical for CSC self-renewal and tumor progression.As a key glycolytic enzyme,hexokinase 2(HK2)plays an instrumental role in aerobic glycolysis and tumor progression.However,whether HK2 directly contribute to CSC stemness maintenance in small cell lung cancer(SCLC)is largely unclear.In this study,we aimed to investgate whether HK2 independent of its glycolytic activity is directly involved in stemness maintenance of CSC in SCLC.Methods:Immunoblotting analyses were conducted to determine the expression of HK2 in SCLC CSCs and their differentiated counterparts.CSC-like properties and tumorigenesis of SCLC cells with or without HK2 depletion or overexpression were examined by sphere formation assay and xenograft mouse model.Immunoprecipitation and mass spectrometry analyses were performed to identify the binding proteins of CD133.The expression levels of CD133-associated and CSC-relevant proteins were evaluated by immunoblotting,immunoprecipitation,immunofluorescence,and immunohistochemistry assay.RNA expression levels of Nanog,POU5F1,Lin28,HK2,Prominin-1 were analyzed through quantitative reverse transcription PCR.Polyubiquitination of CD133 was examined by in vitro or in vivo ubiquitination assay.CD133+cells were sorted by flow cytometry using an anti-CD133 antibody.Results:We demonstrated that HK2 expression was much higher in CSCs of SCLC than in their differentiated counterparts.HK2 depletion inhibited CSC stemness and promoted CSC differentiation.Mechanistically,nonmitochondrial HK2 directly interacted with CD133 and enhanced CD133 expression without affecting CD133 mRNA levels.The interaction of HK2 and CD133 promoted the binding of the deubiquitinase ubiquitin-specific protease 11(USP11)to CD133,thereby inhibiting CD133 polyubiquitylation and degradation.HK2-mediated upregulation of CD133 expression enhanced the expression of cell renewal regulators,SCLC cell stemness,and tumor growth in mice.In addition,HK2 expression was positively correlated with CD133 expression in human SCLC specimens,and their expression levels were associated with poor prognosis of SCLC patients.Conclusions:These results revealed a critical non-metabolic function of HK2 in promotion of cancer cell stemness.Our findings provided new insights into the multifaceted roles of HK2 in tumor development.

USP11通过去泛素化p53调控p53稳定性(英文)

研究目的:深入研究p53的泛素化及稳定性的调控。创新要点:发现一个新的调控p53去泛素化的酶USP11,它可以通过与p53的结合去泛素化并稳定p53,从而揭示了一个新的p53去泛素化调控的机制。研究方法:通过免疫共沉淀发现p53可以与USP11结合(图1a),通过泛素化检测试验发现USP11可以去泛素化p53(图3a和3b),最后通过逆转录-聚合酶链式反应(RT-PCR)试验发现在DNA损伤后,USP11对p53转录活性的提高是非常重要的。重要结论:USP11可通过去泛素化p53来调控p53稳定性。