DNA helicase PIF1 regulates cell proliferation and DNA damage in ovarian cancer
10.16016/j.2097-0927.202408106
- VernacularTitle:DNA解旋酶PIF1调控卵巢癌细胞增殖和DNA损伤
- Author:
Qiyin ZHOU
1
;
Jialin GUO
;
Qingqing SUN
;
Xiaomin WANG
;
Ziwei HU
;
Weiwei PAN
Author Information
1. 310053 杭州,浙江中医药大学基础医学院
- Keywords:
ovarian canser;
PIF1;
DNA damage;
drug resistance;
Rad51 recombinase
- From:
Journal of Army Medical University
2024;46(24):2707-2722
- CountryChina
- Language:Chinese
-
Abstract:
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 a β-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 clonogenic ability(P＜0.001)of ovarian cancer cells,which was also validated in mouse model(P＜0.05).Flow cytometry indicated that PIF1 knockout promoted apoptosis(P＜0.01)and induced cell cycle arrest(P＜0.01)in ovarian cancer cells.In addition,β-galactosidase activity assay demonstrated that PIF1 knockout enhanced cellular senescence(P＜0.001).Western blot and CCK-8 assays further revealed that PIF1 knockout increased the expression of γ-H2AX protein(P＜0.05)and suppressed the proliferative capacity of ovarian cancer cells following cisplatin treatment(P＜0.05).In PIF1 knockout ovarian cancer cells,Rad51 expression was diminished.However,overexpression of Rad51 in PIF1-deficient cells restored PIF1 expression,decreased γ-H2AX protein level(P＜0.05),and rescued cell proliferation(P＜0.01).Immunofluorescence assay demonstrated that EGFP-PIF1 and Rad51 were co-localized in the nucleus.Conclusion PIF1 and Rad51 collaboratively regulate DNA damage and cell proliferation in ovarian cancer cells.
