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目的在细胞水平上分析新发现的DNA损伤修复因子WDR70的生物学功能,确定WDR70基因在人卵巢癌中的突变发生情况,以验证该基因的功能丢失是否与卵巢癌关联。方法在人细胞中用siRNA干扰WDR70基因表达,或用慢病毒和质粒过表达WDR70的野生型和突变体,通过免疫印迹和免疫荧光等方法研究该基因在DNA损伤后的亚细胞定位和DNA损伤信号通路中的作用;此外,抽提1例正常卵巢组织和16例卵巢癌标本的mRNA进行半定量RT-PCR扩增,对这些标本中的WDR70基因进行测序分析。结果 WDR70基因沉默和过表达其突变体导致同源重组功能蛋白——DNA复制蛋白A(RPA32)磷酸化修饰水平降低和重组酶——重组蛋白A(RAD51)向DNA损伤位点招募的能力减低;WDR70的功能障碍还导致染色体的断裂增多;同时,卵巢癌样本中发现多例WDR70突变型别。结论在体外系统中,WDR70参与DNA损伤修复过程,沉默或过表达其突变体将导致同源重组修复缺陷和染色体结构的不稳定;在卵巢癌基因组中,WDR70基因频繁出现突变,可能导致相应的DNA修复缺陷和基因组不稳定性的发生。因此,WDR70是一个潜在的卵巢癌抗癌基因。
OBJECTIVE: To analyze the biological function of newly discovered DNA damage repair factor WDR70 at the cellular level to determine the mutation of WDR70 gene in human ovarian cancer and to verify whether the loss of function of this gene is associated with ovarian cancer. Methods WDR70 gene expression was induced in human cells by siRNA or the wild type and mutant of WDR70 were overexpressed by lentivirus and plasmid. The subcellular localization and DNA damage of the gene after DNA damage were studied by Western blot and immunofluorescence Signal pathway. In addition, mRNA of a normal ovarian tissue and 16 ovarian cancers was extracted for semi-quantitative RT-PCR amplification. The WDR70 gene in these samples was sequenced. Results Silencing and overexpression of WDR70 gene resulted in the decrease of phosphorylation level of homologous recombination function protein - DNA replication protein A (RPA32) and the ability of recombinase - recombinant protein A (RAD51) to recruit to DNA damage site ; Dysfunction of WDR70 also led to increased chromosome rupture; the same time, multiple samples of ovarian cancer found WDR70 mutant type. Conclusion In vitro, WDR70 is involved in the process of DNA damage repair. Silencing or overexpression of its mutant will result in defects of homologous recombination repair and instability of chromosome structure. Frequent mutations of WDR70 gene in ovarian cancer genome may lead to the corresponding DNA repair defects and genomic instability. Therefore, WDR70 is a potential ovarian cancer anti-cancer gene.