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

Qiyin ZHOU; Jialin GUO; Qingqing SUN; Xiaomin WANG; Ziwei HU; Weiwei PAN

doi:10.16016/j.2097-0927.202408106

您当前的位置：

首页 >

文章列表页 >

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

Basic Medicine | 更新时间：2026-01-09

- DNA helicase PIF1 regulates cell proliferation and DNA damage in ovarian cancer
- Journal of Army Medical University Vol. 46, Issue 24, Pages: 2707-2722(2024)
- 作者机构：
  
  1.310053 杭州，浙江中医药大学基础医学院
  2.314001 浙江嘉兴，嘉兴大学医学院
- 作者简介：
- 基金信息：
  
  the General Program of National Natural Science Foundation of China(31871402);the Natural Science Foundation of Zhejiang Province(LGD22H030004);the Pioneer Innovation Team Project of Innovative Jiaxing·Talent Support Plan(2021-08)
- DOI：10.16016/j.2097-0927.202408106
  CLC： R345;R730.23;R737.31
- Received：25 August 2024，
  
  Revised：2024-09-05，
  
  Published：30 December 2024
- 稿件说明：
移动端阅览
Qiyin ZHOU, Jialin GUO, Qingqing SUN, et al. DNA helicase PIF1 regulates cell proliferation and DNA damage in ovarian cancer[J]. Journal of Army Medical University, 2024, 46(24): 2707-2722.
DOI：

Qiyin ZHOU, Jialin GUO, Qingqing SUN, et al. DNA helicase PIF1 regulates cell proliferation and DNA damage in ovarian cancer[J]. Journal of Army Medical University, 2024, 46(24): 2707-2722. DOI： 10.16016/j.2097-0927.202408106.

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

PIF

1基因在正常卵巢组织和卵巢癌组织中的相对表达量

以及

PIF

1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(

=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

PIF

1过表达与患者的总体生存率呈负相关(

0.001);且相比较正常卵巢组织

PIF

1在卵巢癌组织中过表达(

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(

0.01)和克隆能力(

0.001)

且在小鼠体内也得到验证(

0.05)。流式细胞术显示

PIF1敲除促进卵巢癌细胞凋亡(

0.01)

阻滞细胞周期(

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(

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(

0.05)

并且抑制顺铂处理后的卵巢癌细胞的增殖能力(

0.05)。在PIF1敲除的卵巢癌细胞中

Rad51表达减少。而在PIF1缺失的细胞中过表达Rad51蛋白后PIF1表达增加

γ-H2AX蛋白表达减少(

0.05)

并且挽救细胞增殖(

0.01)。免疫荧光显示

EGFP-PIF1和Rad51在细胞核内共定位。

结论

PIF1和Rad51共同调控卵巢癌细胞的DNA损伤和细胞增殖。

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

PIF

1 gene in normal ovarian tissue compared to ovarian cancer tissue

as well as the relationship between

PIF

1 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 u

sed 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

PIF

1 overexpression was negatively correlated with the overall survival of patients (

0.001)

and

PIF

1 was found to be overexpressed in the ovarian cancer tissues than the normal ovarian tissues (

0.05).CRISPR/Cas9-mediated knockout of PIF1 significantly inhibited the proliferation (

0.01) and clonogenic ability (

0.001) of ovarian cancer cells

which was also validated in mouse model (

0.05).Flow cytometry indicated that PIF1 knockout promoted apoptosis (

0.01) and induced cell cycle arrest (

0.01) in ovarian cancer cells.In addition

β-galactosidase activity assay demonstrated that PIF1 knockout enhanced cellular senescence (

0.001).Western blot and CCK-8 assays further revealed that PIF1 knockout increased the expression of γ-H2AX prote

in (

0.05) and suppressed the proliferative capacity of ovarian cancer cells following cisplatin treatment (

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 (

0.05)

and rescued cell proliferation (

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.

关键词

Keywords

references

TORRE L A, TRABERT B, DESANTIS C E, et al . Ovarian cancer statistics, 2018[J ] . CA Cancer J Clin, 2018, 68(4): 284-296. DOI: 10.3322/caac.21456.

ALATISE K L, GARDNER S, ALEXANDER-BRYANT A. Mechanisms of drug resistance in ovarian cancer and associated gene targets[J]. Cancers, 2022, 14(24): 6246. DOI: 10.3390/cancers14246246.

DAMIA G, BROGGINI M. Platinum resistance in ovarian cancer: role of DNA repair[J]. Cancers, 2019, 11(1): 119. DOI: 10.3390/cancers11010119.

BOUBERHAN S, BAR-PELED L, MATOBA Y, et al . The evolving role of DNA damage response in overcoming therapeutic resistance in ovarian cancer[J ] . Cancer Drug Resist, 2023, 6(2): 345-357. DOI: 10.20517/cdr.2022.146.

MURAI J. Targeting DNA repair and replication stress in the treatment of ovarian cancer[J]. Int J Clin Oncol, 2017, 22(4): 619-628. DOI: 10.1007/s10147-017-1145-7.

DING L, LIU Y L. Borrowing nuclear DNA helicases to protect mitochondrial DNA[J]. Int J Mol Sci, 2015, 16(5): 10870-10887. DOI: 10.3390/ijms160510870.

MALONE E G, THOMPSON M D, BYRD A K. Role and regulation of Pif1 family helicases at the replication fork[J]. Int J Mol Sci, 2022, 23(7): 3736. DOI: 10.3390/ijms23073736.

WILSON M A, KWON Y, XU Y Y, et al . Pif1 helicase and Polδ promote recombination-coupled DNA synthesis via bubble migration[J ] . Nature, 2013, 502(7471): 393-396. DOI: 10.1038/nature12585..

LAHAYE A, STAHL H, THINES-SEMPOUX D, et al . PIF1: a DNA helicase in yeast mitochondria[J ] . EMBO J, 1991, 10(4): 997-1007. DOI: 10.1002/j.1460-2075.1991.tb08034.x.

JIMENO S, CAMARILLO R, MEJIAS-NAVARRO F, et al . The helicase PIF1 facilitates resection over sequences prone to forming G4 structures[J ] . Cell Rep, 2018, 25(12): 3543. DOI: 10.1016/j.celrep.2018.12.029.

ZHOU J, MONSON E K, TENG S C, et al . Pif1p helicase, a catalytic inhibitor of telomerase in yeast[J ] . Science, 2000, 289(5480): 771-774.DOI: 10.1126/science.289.5480.771.

MCDONALD K R, SABOURI N, WEBB C J, et al . The Pif1 family helicase Pfh1 facilitates telomere replication and has an RPA-dependent role during telomere lengthening[J ] . DNA Repair, 2014, 24: 80-86. DOI: 10.1016/j.dnarep.2014.09.008.

GERONIMO C L, ZAKIAN V A. Getting it done at the ends: Pif1 family DNA helicases and telomeres[J]. DNA Repair, 2016, 44: 151-158. DOI: 10.1016/j.dnarep.2016.05.021.

WANG J C, ZHU X Y, YING P, et al . PIF1 affects the proliferation and apoptosis of cervical cancer cells by influencing TERT[J ] . Cancer Manag Res, 2020, 12: 7827-7835. DOI: 10.2147/CMAR.S265336.

CAI T, WANG N, MENG P , et al . Up-regulated PIF1 predicts poor clinical outcomes and correlates with low immune infiltrates in clear cell renal cell carcinoma[J ] . Front Genet, 2022, 13: 1058040.DOI: 10.3389/fgene.2022.1058040.

WANG K, HUA X D, FU X B, et al . Petite integration factor 1 knockdown enhances gemcitabine sensitivity in pancreatic cancer cells via increasing DNA damage[J ] . J Appl Toxicol, 2023, 43(10): 1522-1532. DOI: 10.1002/jat.4494..

CHEN B, HUA Z Y, GONGB C, et al . Downregulation of PIF1, a potential new target of MYCN, induces apoptosis and inhibits cell migration in neuroblastoma cells[J ] . Life Sci, 2020, 256: 117820. DOI: 10.1016/j.lfs.2020.117820.

ZHANG T, WU D M, LUO P W, et al . CircNEIL3 mediates pyroptosis to influence lung adenocarcinoma radiotherapy by upregulating PIF1 through miR-1184 inhibition[J ] . Cell Death Dis, 2022, 13(2): 167. DOI: 10.1038/s41419-022-04561-x.

GAGOU M E, GANESH A, THOMPSON R, et al . Suppression of apoptosis by PIF1 helicase in human tumor cells[J ] . Cancer Res, 2011, 71(14): 4998-5008. DOI: 10.1158/0008-5472.CAN-10-4404.

LI S B, WANG H L, JEHI S, et al . PIF1 helicase promotes break-induced replication in mammalian cells[J ] . EMBO J, 2021, 40(8): e104509. DOI: 10.15252/embj.2020104509.

EVAN G I, VOUSDEN K H. Proliferation, cell cycle and apoptosis in cancer[J]. Nature, 2001, 411(6835): 342-348. DOI: 10.1038/35077213.

CALCINOTTO A, KOHLI J, ZAGATO E, et al . Cellular senescence: aging, cancer, and injury[J ] . Physiol Rev, 2019, 99(2): 1047-1078. DOI: 10.1152/physrev.00020.2018.

MATEYAK M K, ZAKIAN V A. Human PIF helicase is cell cycle regulated and associates with telomerase[J]. Cell Cycle, 2006, 5(23): 2796-2804. DOI: 10.4161/cc.5.23.3524.

FUTAMI K, SHIMAMOTO A, FURUICHI Y. Mitochondrial and nuclear localization of human Pif1 helicase[J]. Biol Pharm Bull, 2007, 30(9): 1685-1692. DOI: 10.1248/bpb.30.1685.

ONONYE O E, SAUSEN C W, BALAKRISHNAN L, et al . Lysine acetylation regulates the activity of nuclear Pif1[J ] . J Biol Chem, 2020, 295(46): 15482-15497. DOI: 10.1074/jbc.RA120.015164.

ZOU L E, ELLEDGE S J. Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes[J]. Science, 2003, 300(5625): 1542-1548. DOI: 10.1126/science.1083430.

JIANG K C, PEREIRA E, MAXFIELD M, et al . Regulation of Chk1 includes chromatin association and 14-3-3 binding following phosphorylation on Ser -345[J ] . J Biol Chem, 2003, 278(27): 25207-25217. DOI: 10.1074/jbc.M300070200..

MARTIN S A, OUCHI T. Cellular commitment to reentry into the cell cycle after stalled DNA is determined by site-specific phosphorylation of Chk1 and PTEN[J]. Mol Cancer Ther, 2008, 7(8): 2509-2516. DOI: 10.1158/1535-7163.MCT-08-0199.

FREEDMAN D A, WU L, LEVINE A J. Functions of the MDM2 oncoprotein[J]. Cell Mol Life Sci, 1999, 55(1): 96-107. DOI: 10.1007/s000180050273.

JIN M H, OH D Y. ATM in DNA repair in cancer[J]. Pharmacol Ther, 2019, 203: 107391. DOI: 10.1016/j.pharmthera.2019.07.002.

WRIGHT W D, SHAH S S, HEYER W D. Homologous recombination and the repair of DNA double-strand breaks[J]. J Biol Chem, 2018, 293(27): 10524-10535. DOI: 10.1074/jbc.TM118.000372.

LIU L P, MALKOVA A. Break-induced replication: unraveling each step[J]. Trends Genet, 2022, 38(7): 752-765. DOI: 10.1016/j.tig.2022.03.011.

Views

下载量

CSCD

Alert me when the article has been cited

提交

Tools

Publicity Resources

TAS102 selectively inhibits proliferation of α-thalassemia mental retardation X-linked gene-deficient glioma cells

Myocardial-specific Hdac3 deletion induces by ventricular remodeling in mice

Trichostatin A enhances cisplatin-induced cytotoxicity through inhibiting base excision repair of AP endonuclease in lung adenocarcinoma cells

Targeting DNA repair pathways: a new episode in targeted cancer therapeutics

XPC protein promotes UV DNA damage response of human bladder cancer cells

Related Author

Yundong ZHANG

Fei LI

Hua FENG

Ning MU

Suna HUANG

Xuegang LI

Tunan CHEN

Hang JIANG

Related Institution

Department of Neurological Diseases, the Third Affiliated Hospital of Chongqing Medical University

Department of Neurosurgery, First Affiliated Hospital, Army Medical University(Third Military Medical University)

Department of Cardiology, the Second Affiliated Hospital of Chongqing Medical University

Department of Pathology, West China Hospital, Sichuan University

⁰