唐子执, 王海斌, 曾 鸣, 等.CRL4泛素化酶在卵巢癌中的功能和突变研究.四川大学学报(医学版),2017,48(5):693-698
CRL4泛素化酶在卵巢癌中的功能和突变研究
DNA Repair Function and Mutation of an H2B Monoubiquitination Factor WDR70 in Ovarian Cancer
  
中文关键词:  H2B单泛素化 CRL4 DNA损伤应答 卵巢癌
英文关键词:H2B monoubiquitination CRL4 DNA damage responses Ovarian cancer
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中文摘要:
      目的 研究泛素化酶CRL4蛋白复合体家族成员CRL4WD40重复序列结构域蛋白70(WDR70)在卵巢癌细胞中的DNA修复功能,以及卵巢癌组织中该泛素化酶基因的突变规律。方法 利用免疫荧光方法,检测CRL4骨架蛋白DDB1及WDR70基因特异性沉默的卵巢癌细胞与其对应的对照组细胞在化疗药物或放射线照射诱导产生DNA双链断裂后,组蛋白H2AX(γH2AX)及单链DNA结合蛋白32(RPA32)磷酸化灶点显示的差异;BrdU标记和染色实验检测WDR70基因对DNA复制是否存在影响,同时利用免疫组化染色检测卵巢癌组织临床病理标本及正常卵巢组织标本中的WDR70和组蛋白H2B单泛素化(uH2B)染色差异,以阐明CRL4的DNA损伤应答特征,RTPCR测定卵巢癌组织中WDR70的基因表达水平,并采用DNA测序确定WDR70突变位点。结果 免疫荧光染色结果显示,CRL4WDR70的不同蛋白亚基(DDB1、WDR70)在细胞周期检验点激活和uH2B介导的DNA末端回切过程中起着不同的作用:DDB1参与以上两个机制的调控,而WDR70只促进末端回切、RPA32在DNA断裂点的招募和同源重组修复。BrdU标记和染色结果显示WDR70基因对DNA复制并不存在影响。免疫组化结果显示,卵巢癌组织临床病理标本及正常卵巢组织标本中的WDR70和uH2B表达存在差异。RTPCR结果显示WDR70基因的全长、5′和3′转录本水平在50%的卵巢癌组织中水平减低,出现多处外显子突变位点。结论 CRL4在DNA修复过程中具有促进H2B单泛素化、促进DNA末端回切和激活细胞周期检验点等多种重要功能,是维持基因组稳定性、遏阻卵巢癌发生的重要抗癌机制。
英文摘要:
      【Abstract】 Objective To investigate the roles of enzyme DCAF proteinDNA damagebinding protein 1 (DDB1)/cullin4 (CRL4) complex family members CRL4WD40 repeat domain protein 70 (WDR70) in DNA repair process and its mutation in ovarian cancer. Methods Immunofluorescent assay was employed to measure H2AX(γH2AX) and phosphorylated replication protein A2 (RPA32) formed in siDDB1 or siWDR70 ovarian cancer cells after the treatments of chemical medicine and radioactive threapy. 5Brdu immunohistochemical staining was used to explore the function of WDR70 in DNA replication. The expressions of WDR70 and histone protein H2B monoubiquitination (uH2B) was measured by immunohistochemistry, the function of DNA repair, expression and mutations of CRL4 in ovarian cancer were detected by semiquantitative PCR and DNA sequencing. Results Immunofluorescent assay indicated that distinct subunits of CRL4 played different roles in checkpoint activation and H2Bmonoubiquitinationdepedendent homologous recombination, while the scaffold subunit DDB1 participated in both processes, WDR70 was only required for DNA end resection, chromatin loading of RPA32 and HR. The dose of WDR70 was not effect on DNA replication. Ovarian cancer had different expression of WDR70 and uH2B compared with normal tissue, transcripts of WDR70 was diminished or truncated in 50% of ovarian cancer, which corresponded to multiple mutations. Conclusion CRL4 ubiquitin ligase plays multiple roles in DNA repair and is critical for genome stability. It may be an potential anticancer barrier against ovarian malignancies.
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