ZW10相互作用着丝粒蛋白对神经母细胞瘤增殖的影响及机制研究  

The effect and mechanism of ZW10 interacting kinetochore protein on proliferation of neuroblastoma cells

作　　者：袁文 蔡秦真 王军[1] 庹文彬 向贇[1] 袁纯辉 YUAN Wen;CAI Qinzhen;WANG Jun;TUO Wenbin;XIANG Yun;YUAN Chunhui(Department of Laboratory Medicine,Wuhan Children's Hospital(Wuhan Maternal and Child Healthcare Hospital),Tongji Medical College,Huazhong University of Science&Technology,Hubei Wuhan 430016,China)

机构地区：[1]华中科技大学同济医学院附属武汉儿童医院(武汉市妇幼保健院)检验科,湖北武汉430016

出　　处：《现代肿瘤医学》2023年第1期43-50,共8页Journal of Modern Oncology

基　　金：湖北省卫健委科研项目(编号:WJ2021M016);湖北省武汉市卫健委科研项目(编号:WX20C03,WX18Q03)。

摘　　要：目的:探讨ZW10相互作用着丝粒蛋白(ZW10 interacting kinetochore protein,ZWINT)对神经母细胞瘤(neuroblastoma,NB)细胞增殖的影响及作用机制。方法:利用NB数据集分析ZWINT表达水平与NB预后及临床分期的关系。EdU实验、流式细胞术和γH2AX免疫荧光染色分别检测干扰ZWINT表达对NB细胞增殖、周期分布和DNA损伤修复能力的影响。免疫组织化学/蛋白质印记(Western blot)检测MYCN扩增和无扩增组织及细胞系中ZWINT的表达情况。荧光定量PCR(qRT-PCR)和Western blot检测干扰/过表达MYCN对ZWINT表达的影响。在线预测ZWINT的启动子区域MYCN的结合位点,ChIP-PCR和荧光素酶实验验证MYCN靶向调控ZWINT表达。强力霉素诱导Tet-on MYCN SH-SY-5Y细胞过表达MYCN同时干扰ZWINT表达,检测ZWINT对MYCN介导的细胞增殖、周期和DNA损伤修复功能的影响。结果:ZWINT表达水平与NB患儿总生存率、无事件生存率呈负相关,其表达随NB分期升高而增高。ZWINT干扰组NB细胞增殖明显减少,G_(2)/M期细胞比例显著增高,γH2AX焦点的形成增加。ZWINT在MYCN扩增的NB组织/细胞系中表达水平显著高于无扩增组。MYCN与ZWINT启动子区结合,干扰ZWINT表达可显著抑制MYCN对增殖的促进作用。结论:ZWINT是NB的不良预后指标,其表达受MYCN调控,干扰ZWINT表达导致MYCN扩增的NB细胞周期阻滞在G2/M期,DNA损伤修复增加,抑制NB细胞的增殖。Objective:To investigate the effect of ZW10 interacting kinetochore protein(ZWINT)on neuroblastoma(NB)cell proliferation and its potential mechanism.Methods:GEO database was used to analyze the correlation of ZWINT with prognosis and different clinical stages in NB.ZWINT was interfered in MYCN amplified NB cell lines by shRNA,and cell proliferation was then evaluated by using EdU cell proliferation assay.Cell cycle distribution was detected by flow cytometry.The repair ability of DNA damage was detected byγH2 AX immunofluorescence staining.The effect of interfering/overexpressing MYCN on ZWINT expression was analyzed by qRT-PCR and Western blot.The binding site of MYCN on ZWINT promoter was predicted by online database,and the effect of MYCN on ZWINT transcription was detected by ChIP-PCR and luciferase assay.To further validate the effects of ZWINT on MYCN-mediated cell proliferation,cycle and DNA damage repair,we examined Tet-on MYCN SH-SY-5 Y cells with ZWINT knockdown.Results:High expression of ZWINT mRNA was related to significantly shorter overall and event-free survival for neuroblastoma patients.Moreover,increased ZWINT expression correlated significantly with advanced tumor stages.ZWINT silencing efficiently inhibited cellular proliferation,increased the percentage of cell population in G_(2)/M phase,and increased the formation ofγH2 AX foci.ZWINT expression of MYCN-amplified was significantly higher than non-MYCNN-amplified NB tissues and cell lines.MYCN can bind to promoter and regulate ZWINT expression.Overexpression of MYCN significantly promoted SH-SY-5 Y cells proliferation,and which could be blocked by simultaneously transfecting with shZWINT.Conclusion:ZWINT is a prognostic marker in NB,which was regulated by MYCN.Silencing ZWINT induced G_(2)/M cell cycle arrest,and DNA damage repair increase,thereby inhibited the proliferation of MYCN-amplified NB cell.

关 键 词：神经母细胞瘤 ZW10相互作用着丝粒蛋白 MYCN扩增 增殖 细胞周期 

分 类 号：R730.264[医药卫生—肿瘤]