Transcriptome Analysis of FEN1 Knockdown HEK293T Cell Strain Reveals Alteration in Nucleic Acid Metabolism, Virus Infection, Cell Morphogenesis and Cancer Development

Author(s): Song-Bai Liu*, Xiu-Qin Qiu, Wei-Qiang Guo, Jin-Li Li, Qian Su, Jia-Hui Du, He-Juan Hu, Xiao-Xiao Wang, Yao-Hua Song, Xiao Lou*, Xiang-Bin Xu*.

Journal Name: Combinatorial Chemistry & High Throughput Screening
Accelerated Technologies for Biotechnology, Bioassays, Medicinal Chemistry and Natural Products Research

Volume 22 , Issue 6 , 2019

Become EABM
Become Reviewer

Abstract:

Aim and Objective: Flap endonuclease-1 (FEN1) plays a central role in DNA replication and DNA damage repair process. In mammals, FEN1 functional sites variation is related to cancer and chronic inflammation, and supports the role of FEN1 as a tumor suppressor. However, FEN1 is overexpressed in multiple types of cancer cells and is associated with drug resistance, supporting its role as an oncogene. Hence, it is vital to explore the multi-functions of FEN1 in normal cell metabolic process. This study was undertaken to examine how the gene expression profile changes when FEN1 is downregulated in 293T cells.

Materials and Methods: Using the RNA sequencing and real-time PCR approaches, the transcript expression profile of FEN1 knockdown HEK293T cells have been detected for the next step evaluation, analyzation, and validation.

Results: Our results confirmed that FEN1 is important for cell viability. We showed that when FEN1 downregulation led to the interruption of nucleic acids related metabolisms, cell cycle related metabolisms are significantly interrupted. FEN1 may also participate in non-coding RNA processing, ribosome RNA processing, transfer RNA processing, ribosome biogenesis, virus infection and cell morphogenesis.

Conclusion: These findings provide insight into how FEN1 nuclease might regulate a wide variety of biological processes, and laid the foundation for understanding the role of other RAD2 family nucleases in cell growth and metabolism.

Keywords: FEN1, DNA replication, RNA sequencing, RNA process, cancer, nucleic acid metabolism.

[1]
Lee, B.I.; Wilson, D.M., III The RAD2 domain of human exonuclease 1 exhibits 5′ to 3′ exonuclease and flap structure-specific endonuclease activities. J. Biol. Chem., 1999, 274(53), 37763-37769.
[http://dx.doi.org/10.1074/jbc.274.53.37763] [PMID: 10608837]
[2]
Shen, B.; Singh, P.; Liu, R.; Qiu, J.; Zheng, L.; Finger, L.D.; Alas, S. Multiple but dissectible functions of FEN-1 nucleases in nucleic acid processing, genome stability and diseases. BioEssays, 2005, 27(7), 717-729.
[http://dx.doi.org/10.1002/bies.20255] [PMID: 15954100]
[3]
Lieber, M.R. The FEN-1 family of structure-specific nucleases in eukaryotic DNA replication, recombination and repair. BioEssays, 1997, 19(3), 233-240.
[http://dx.doi.org/10.1002/bies.950190309] [PMID: 9080773]
[4]
Harrington, J.J.; Lieber, M.R. Functional domains within FEN-1 and RAD2 define a family of structure-specific endonucleases: implications for nucleotide excision repair. Genes Dev., 1994, 8(11), 1344-1355.
[http://dx.doi.org/10.1101/gad.8.11.1344] [PMID: 7926735]
[5]
Guo, Z.; Qian, L.; Liu, R.; Dai, H.; Zhou, M.; Zheng, L.; Shen, B. Nucleolar localization and dynamic roles of flap endonuclease 1 in ribosomal DNA replication and damage repair. Mol. Cell. Biol., 2008, 28(13), 4310-4319.
[http://dx.doi.org/10.1128/MCB.00200-08] [PMID: 18443037]
[6]
Liu, S.; Lu, G.; Ali, S.; Liu, W.; Zheng, L.; Dai, H.; Li, H.; Xu, H.; Hua, Y.; Zhou, Y.; Ortega, J.; Li, G.M.; Kunkel, T.A.; Shen, B. Okazaki fragment maturation involves α-segment error editing by the mammalian FEN1/MutSα functional complex. EMBO J., 2015, 34(13), 1829-1843.
[http://dx.doi.org/10.15252/embj.201489865] [PMID: 25921062]
[7]
Balakrishnan, L.; Bambara, R.A. Flap endonuclease 1. Annu. Rev. Biochem., 2013, 82, 119-138.
[http://dx.doi.org/10.1146/annurev-biochem-072511-122603] [PMID: 23451868]
[8]
Asagoshi, K.; Tano, K.; Chastain, P.D., II; Adachi, N.; Sonoda, E.; Kikuchi, K.; Koyama, H.; Nagata, K.; Kaufman, D.G.; Takeda, S.; Wilson, S.H.; Watanabe, M.; Swenberg, J.A.; Nakamura, J. FEN1 functions in long patch base excision repair under conditions of oxidative stress in vertebrate cells. Mol. Cancer Res., 2010, 8(2), 204-215.
[http://dx.doi.org/10.1158/1541-7786.MCR-09-0253] [PMID: 20145043]
[9]
Scott, T.L.; Rangaswamy, S.; Wicker, C.A.; Izumi, T. Repair of oxidative DNA damage and cancer: Recent progress in DNA baseexcision repair. Antioxid. Redox Signal., 2014, 20(4), 708-726.
[10]
Frosina, G.; Fortini, P.; Rossi, O.; Carrozzino, F.; Raspaglio, G.; Cox, L.S.; Lane, D.P.; Abbondandolo, A.; Dogliotti, E. Two pathways for base excision repair in mammalian cells. J. Biol. Chem., 1996, 271(16), 9573-9578.
[http://dx.doi.org/10.1074/jbc.271.16.9573] [PMID: 8621631]
[11]
Larsen, E.; Gran, C.; Saether, B.E.; Seeberg, E.; Klungland, A. Proliferation failure and gamma radiation sensitivity of FEN1 null mutant mice at the blastocyst stage. Mol. Cell. Biol., 2003, 23(15), 5346-5353.
[http://dx.doi.org/10.1128/MCB.23.15.5346-5353.2003] [PMID: 12861020]
[12]
Sun, H.; He, L.; Wu, H.; Pan, F.; Wu, X.; Zhao, J.; Hu, Z.; Sekhar, C.; Li, H.; Zheng, L.; Chen, H.; Shen, B.H.; Guo, Z. The FEN1 L209P mutation interferes with long-patch base excision repair and induces cellular transformation. Oncogene, 2017, 36(2), 194-207.
[13]
Kucherlapati, M.; Yang, K.; Kuraguchi, M.; Zhao, J.; Lia, M.; Heyer, J.; Kane, M.F.; Fan, K.; Russell, R.; Brown, A.M.; Kneitz, B.; Edelmann, W.; Kolodner, R.D.; Lipkin, M.; Kucherlapati, R. Haploinsufficiency of Flap endonuclease (FEN1) leads to rapid tumor progression. Proc. Natl. Acad. Sci. USA, 2002, 99(15), 9924-9929.
[http://dx.doi.org/10.1073/pnas.152321699] [PMID: 12119409]
[14]
Zheng, L.; Dai, H.; Zhou, M.; Li, M.; Singh, P.; Qiu, J.; Tsark, W.; Huang, Q.; Kernstine, K.; Zhang, X.; Lin, D.; Shen, B. FEN1 mutations result in autoimmunity, chronic inflammation and cancers. Nat. Med., 2007, 13(7), 812-819.
[http://dx.doi.org/10.1038/nm1599] [PMID: 17589521]
[15]
He, L.; Luo, L.; Zhu, H.; Yang, H.; Zhang, Y.; Wu, H.; Sun, H.; Jiang, F.; Kathera, C.S.; Liu, L.; Zhuang, Z.; Chen, H.; Pan, F.; Hu, Z.; Zhang, J.; Guo, Z. FEN1 promotes tumor progression and confers cisplatin resistance in non-small-cell lung cancer. Mol. Oncol., 2017, 11(6), 640-654.
[http://dx.doi.org/10.1002/1878-0261.12058] [PMID: 28371273]
[16]
Wang, Z.; Gerstein, M.; Snyder, M. RNA-Seq: A revolutionary tool for transcriptomics. Nat. Rev. Genet., 2009, 10(1), 57-63.
[http://dx.doi.org/10.1038/nrg2484] [PMID: 19015660]
[17]
Burcham, P.C. Internal hazards: baseline DNA damage by endogenous products of normal metabolism. Mutat. Res., 1999, 443(1-2), 11-36.
[http://dx.doi.org/10.1016/S1383-5742(99)00008-3] [PMID: 10415429]
[18]
Hakem, R. DNA-damage repair; the good, the bad, and the ugly. EMBO J., 2008, 27(4), 589-605.
[http://dx.doi.org/10.1038/emboj.2008.15] [PMID: 18285820]
[19]
Matsunaga, T.; Mu, D.; Park, C.H.; Reardon, J.T.; Sancar, A. Human DNA repair excision nuclease. Analysis of the roles of the subunits involved in dual incisions by using anti-XPG and anti-ERCC1 antibodies. J. Biol. Chem., 1995, 270(35), 20862-20869.
[http://dx.doi.org/10.1074/jbc.270.35.20862] [PMID: 7657672]
[20]
Shibata, A.; Moiani, D.; Arvai, A.S.; Perry, J.; Harding, S.M.; Genois, M.M.; Maity, R.; van Rossum-Fikkert, S.; Kertokalio, A.; Romoli, F.; Ismail, A.; Ismalaj, E.; Petricci, E.; Neale, M.J.; Bristow, R.G.; Masson, J.Y.; Wyman, C.; Jeggo, P.A.; Tainer, J.A. DNA double-strand break repair pathway choice is directed by distinct MRE11 nuclease activities. Mol. Cell, 2014, 53(1), 7-18.
[http://dx.doi.org/10.1016/j.molcel.2013.11.003] [PMID: 24316220]
[21]
Otto, C.J.; Almqvist, E.; Hayden, M.R.; Andrew, S.E. The “flap” endonuclease gene FEN1 is excluded as a candidate gene implicated in the CAG repeat expansion underlying Huntington disease. Clin. Genet., 2001, 59(2), 122-127.
[http://dx.doi.org/10.1034/j.1399-0004.2001.590210.x] [PMID: 11260214]
[22]
Liu, Y.; Zhang, H.; Veeraraghavan, J.; Bambara, R.A.; Freudenreich, C.H. Saccharomyces cerevisiae flap endonuclease 1 uses flap equilibration to maintain triplet repeat stability. Mol. Cell. Biol., 2004, 24(9), 4049-4064.
[http://dx.doi.org/10.1128/MCB.24.9.4049-4064.2004] [PMID: 15082797]
[23]
Singh, P.; Zheng, L.; Chavez, V.; Qiu, J.; Shen, B. Concerted action of exonuclease and Gap-dependent endonuclease activities of FEN-1 contributes to the resolution of triplet repeat sequences (CTG)n- and (GAA)n-derived secondary structures formed during maturation of Okazaki fragments. J. Biol. Chem., 2007, 282(6), 3465-3477.
[http://dx.doi.org/10.1074/jbc.M606582200] [PMID: 17138563]
[24]
Senkevich, T.G.; Koonin, E.V.; Moss, B. Predicted poxvirus FEN1-like nuclease required for homologous recombination, double-strand break repair and full-size genome formation. Proc. Natl. Acad. Sci. USA, 2009, 106(42), 17921-17926.
[http://dx.doi.org/10.1073/pnas.0909529106] [PMID: 19805122]
[25]
Zou, J.; Zhu, L.; Jiang, X.; Wang, Y.; Wang, Y.; Wang, X.; Chen, B. Curcumin increases breast cancer cell sensitivity to cisplatin by decreasing FEN1 expression. Oncotarget, 2018, 9(13), 11268-11278.
[http://dx.doi.org/10.18632/oncotarget.24109] [PMID: 29541412]
[26]
He, L.; Yang, H.; Zhou, S.; Zhu, H.; Mao, H.; Ma, Z.; Wu, T.; Kumar, A.K.; Kathera, C.; Janardhan, A.; Pan, F.; Hu, Z.; Yang, Y.; Luo, L.; Guo, Z. Synergistic antitumor effect of combined paclitaxel with FEN1 inhibitor in cervical cancer cells. DNA Repair (Amst.), 2018, 63, 1-9.
[http://dx.doi.org/10.1016/j.dnarep.2018.01.003] [PMID: 29358095]
[27]
Zeng, X.; Che, X.; Liu, Y.P.; Qu, X.J.; Xu, L.; Zhao, C.Y.; Zheng, C.L.; Hou, K.Z.; Teng, Y. FEN1 knockdown improves trastuzumab sensitivity in human epidermal growth factor 2-positive breast cancer cells. Exp. Ther. Med., 2017, 14(4), 3265-3272.
[http://dx.doi.org/10.3892/etm.2017.4873] [PMID: 28912877]
[28]
Xie, C.; Wang, K.; Chen, D. Flap endonuclease 1 silencing is associated with increasing the cisplatin sensitivity of SGC 7901 gastric cancer cells. Mol. Med. Rep., 2016, 13(1), 386-392.
[http://dx.doi.org/10.3892/mmr.2015.4567] [PMID: 26718738]
[29]
Wang, J.; Zhou, L.; Li, Z.; Zhang, T.; Liu, W.; Liu, Z.; Yuan, Y.C.; Su, F.; Xu, L.; Wang, Y.; Zhou, X.; Xu, H.; Hua, Y.; Wang, Y.J.; Zheng, L.; Teng, Y.E.; Shen, B. YY1 suppresses FEN1 over-expression and drug resistance in breast cancer. BMC Cancer, 2015, 15, 50.
[http://dx.doi.org/10.1186/s12885-015-1043-1] [PMID: 25885449]
[30]
Bray, F.; Ferlay, J.; Soerjomataram, I.; Siegel, R.L.; Torre, L.A.; Jemal, A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin., 2018, 68(6), 394-424.
[http://dx.doi.org/10.3322/caac.21492] [PMID: 30207593]


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Article Details

VOLUME: 22
ISSUE: 6
Year: 2019
Page: [379 - 386]
Pages: 8
DOI: 10.2174/1386207322666190704095602

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