DNA损伤修复因子WDR70的生物学功能及其在人卵巢癌中的突变研究

Functional Analysis of DNA Damage Repair Factor WDR70 and Its Mutation in Ovarian Cancer

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 cellular function of the newly discovered DNA damage repair factor WDR70,and investigate the mutation in ovarian cancer to verify if function loss of the WDR70 gene was associated with ovarian cancer.Methods The WDR70 gene was silenced by using siRNA technique or overexpressed its wild and mutation type by with lentivirus and plasmid in hunman cells.The subcellular localization and biochemical function of WDR70 was analyzes by indirect immunofluorescence and immunoblotting.The expression level of WDR70 and the mutations of its cDNA was checked with RT-PCR sequencing for 1normal ovarian tissue and 16 ovarian cancer specimen.Results We found gene silencing of WDR70 or overexpression of WDR70 mutation type disrupts the phosphorylation level of homologous recombination functional protein RPA32 and the ability of recruitment at DNA damage site of recombinase RAD51,the loss of function of WDR70 also causes the elevation of the chromosome breakage in metaphase.Meanwhile,we also noticed that the existence of multiple mutations in genomic WDR70 in ovarian cancer specimen.Conclusion Our results defined that in vitro system,WDR70 is a DNA damage repair gene,silencing of WDR70 or overexpression of WDR70 mutation type disrupts homologous recombination and chromosomal instability;the frequent mutations of WDR70 gene in genome of ovarian cancer specimens could also lead to DNA repair defeat and gene instability.Consequently WDR70 gene could represent an anti-cancer mechanism for ovarian cancer.