DNA解旋酶PIF1调控卵巢癌细胞增殖和DNA损伤  

DNA helicase PIF1 regulates cell proliferation and DNA damage in ovarian cancer

作　　者：周琦银 郭佳林 孙晴晴 王晓敏 胡紫薇 潘巍巍 ZHOU Qiyin;GUO Jialin;SUN Qingqing;WANG Xiaomin;HU Ziwei;PAN Weiwei(School of Basic Medicine,Zhejiang Chinese Medical University,Hangzhou,Zhejiang Province,310053;School of Medicine,Jiaxing University,Jiaxing,Zhejiang Province,314001,China)

机构地区：[1]浙江中医药大学基础医学院,杭州310053 [2]嘉兴大学医学院,浙江嘉兴314001

出　　处：《陆军军医大学学报》2024年第24期2707-2722,共16页Journal of Army Medical University

基　　金：国家自然科学基金面上项目(31871402);浙江省自然科学基金(LGD22H030004);“创新嘉兴·优才支持计划”先锋型创新团队资助的课题(2021-08)。

摘　　要：目的探究DNA解旋酶PIF1在卵巢癌细胞增殖和DNA损伤中的作用及机制。方法通过基因表达谱交互式分析平台(Gene Expression Profiling Interactive Analysis,GEPIA)和Kaplan-Meier Plotter公共数据库分析PIF1基因在正常卵巢组织和卵巢癌组织中的相对表达量,以及PIF1表达量和卵巢癌患者总生存率的关系。通过CRISPR/Cas9基因编辑技术建立PIF1敲除的卵巢癌细胞。构建Rad51重组酶(Rad51 recombinase,Rad51)过表达的反转录病毒载体,进行PIF1敲除的ES-2卵巢癌细胞转染。采用Western blot评估PIF1敲除和Rad51过表达的效果;通过细胞计数、克隆形成实验和CCK-8评估细胞的增殖能力。选取32只6~8周龄BALB/c雌性裸鼠(体质量20~25 g),按随机数字表法分为4组(n=8):ES-2对照组、ES-2敲除组、OVCAR-3对照组和OVCAR-3敲除组,建立小鼠荷瘤模型评估卵巢癌细胞在体内的增殖能力。通过流式细胞术评估细胞的凋亡率和细胞周期;采用β-半乳糖苷酶活性检测评估卵巢癌细胞的衰老情况。应用Western blot和免疫荧光检测磷酸化组蛋白H2AX(phosphorylated histone H2AX,γ-H2AX)的变化,评估PIF1敲除以及过表达Rad51对DNA损伤的影响,并检测PIF1和Rad51在卵巢癌细胞中的定位。结果公共数据库分析结果显示,PIF1过表达与患者的总体生存率呈负相关(P<0.001);且相比较正常卵巢组织,PIF1在卵巢癌组织中过表达(P<0.05)。CRISPR/Cas9技术介导的PIF1敲除可显著抑制卵巢癌细胞的增殖(P<0.01)和克隆能力(P<0.001),且在小鼠体内也得到验证(P<0.05)。流式细胞术显示,PIF1敲除促进卵巢癌细胞凋亡(P<0.01),阻滞细胞周期(P<0.01)。此外,β-半乳糖苷酶活性检测结果显示,PIF1敲除促进卵巢癌细胞衰老(P<0.001)。Western blot和CCK-8结果进一步显示,PIF1敲除促进卵巢癌细胞γ-H2AX蛋白表达增加(P<0.05),并且抑制顺铂处理后的卵巢癌细胞的增殖能力(P<0.05)。在PIF1敲除的卵巢癌细胞中,Rad51表达减少Objective To investigate the role and mechanism of the DNA helicase PIF1 in the proliferation of ovarian cancer cells and its response to DNA damage.Methods The relative expression of the PIF1 gene in normal ovarian tissue compared to ovarian cancer tissue,as well as the relationship between PIF1 expression and overall survival in ovarian cancer patients,was analyzed using the Gene Expression Profiling Interactive Analysis(GEPIA)and Kaplan-Meier Plotter public databases.PIF1 knockout ovarian cancer cells were established using CRISPR/Cas9 gene-editing technology.A retroviral vector overexpressing Rad51 recombinase was constructed,and then transfected into PIF1 knockout ES-2 ovarian cancer cells.Western blot analysis was used to determine the effects of PIF1 knockout and Rad51 overexpression in the transfected cells.Cell proliferation was assessed with cell counting,colony formation assay and CCK-8 assay.A total of 32 female BALB/c nude mice(6~8 weeks old,weighing 20~25 g)were randomly divided into ES-2 control group,ES-2 knockout group,OVCAR-3 control group,and OVCAR-3 knockout group,with 8 mice in each group.A mouse xenograft model was established to assess the in vivo proliferative capacity of ovarian cancer cells.Apoptotic rate and cell cycle were assessed using flow cytometry.The senescence of ovarian cancer cells was evaluated through aβ-galactosidase activity assay.Western blotting and immunofluorescence assay were applied to determine the changes in phosphorylated histone H2AX(γ-H2AX)protein were measured with to evaluate the effects of PIF1 knockout and Rad51 overexpression on DNA damage and to observe the localization of PIF1 and Rad51 in ovarian cancer cells.Results The analysis of public databases revealed that PIF1 overexpression was negatively correlated with the overall survival of patients(P<0.001),and PIF1 was found to be overexpressed in the ovarian cancer tissues than the normal ovarian tissues(P<0.05).CRISPR/Cas9-mediated knockout of PIF1 significantly inhibited the proliferation(P<0.01)and c

关 键 词：卵巢癌 PIF1 DNA损伤 抗药性 Rad51重组酶 

分 类 号：R345[医药卫生—基础医学] R730.23R737.31