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目的研究泛素化酶CRL4蛋白复合体家族成员CRL4-WD40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DDB1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,RT-PCR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,CRL4-WDR70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。RT-PCR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。
OBJECTIVE: To study the DNA repair function of CRL4-WD40 repetitive domain protein 70 (WDR70), a member of the ubiquitin CRL4 protein complex family, in ovarian cancer cells and to study the mutation rules of this ubiquitin gene in ovarian cancer. Methods Immunofluorescence assay was used to detect the expression of histone H2AX (γH2AX) and single-stranded RNA (siRNA) in ovarian cancer cell line CRS4-DDB1 and WDR70-specific silencing ovarian cancer cells and their corresponding control cells after DNA double-strand breaks induced by chemotherapeutic drugs or radiation. DNA binding protein 32 (RPA32) phosphorylation foci showed differences; BrdU labeling and staining experiments WDR70 gene detection of DNA replication whether there is an impact, and immunohistochemical staining of ovarian cancer tissue samples and normal ovarian tissue specimens WDR70 and histone H2B mono ubiquitination (uH2B) staining were used to elucidate the DNA damage response characteristics of CRL4. The gene expression of WDR70 in ovarian cancer tissues was determined by RT-PCR and the WDR70 mutation site was determined by DNA sequencing. Results The results of immunofluorescence staining showed that different protein subunits of CRL4-WDR70 (DDB1 and WDR70) played different roles in cell cycle checkpoint activation and uH2B-mediated DNA terminal methylation: DDB1 participates in the above two mechanisms Regulatory, whereas WDR70 only promotes terminal back-cutting, recruitment of RPA32 at the DNA breakpoint and homologous recombination repair. BrdU labeling and staining results showed that WDR70 gene had no effect on DNA replication. Immunohistochemical results showed that the expression of WDR70 and uH2B in ovarian cancer tissue samples and normal ovarian tissue specimens were different. RT-PCR results showed that the full-length, 5 ’and 3’ transcript levels of WDR70 gene were reduced in 50% of ovarian cancer tissues, and multiple exon mutation sites were found. Conclusions CRL4 plays an important role in DNA repair process such as promoting monoubiquitination of H2B, promoting DNA end-reversion, and activating cell cycle checkpoints. CRL4 is an important anticancer mechanism for maintaining genomic stability and preventing ovarian cancer.