Archives of Virology

, Volume 163, Issue 12, pp 3395–3402 | Cite as

CRISPR/Cas9-mediated knockout of HIF-1α gene in epithelioma papulosum cyprini (EPC) cells inhibited apoptosis and viral hemorrhagic septicemia virus (VHSV) growth

  • Min Sun Kim
  • Ki Hong KimEmail author
Brief Report


Hypoxia-inducible factor-1 (HIF-1) is a heterodimer of HIF-1α and HIF-1β, and its key role in the regulation of cellular responses to hypoxia has been well-demonstrated. The participation of HIF-1α in apoptosis has been reported in mammals, however, a little information is available on the role of HIF-1α in the progression of apoptosis in fish. In this study, to know the role of HIF-1α in the apoptosis of fish cells, we produced HIF-1α knockout Epithelioma papulosum cyprini (EPC) cells using a CRISPR/Cas9 vector, and a single cell clone showing a heterozygous insertion/deletion (indel) mutation (one nucleotide insertion and one nucleotide deletion in HIF-1α gene) was chosen for further experiments. To confirm the knockout of HIF-1α, cells were transfected with a hypoxia reporting vector containing hypoxic response elements (HREs). EPC cells transfected with the reporting plasmids showed significantly increased luminescence by exposure to cobalt chloride, a prolyl hydroxylases inhibitor. On the other hand, HIF-1α knockout EPC cells showed a non-responsiveness to a cobalt chloride exposure, suggesting that functional HIF-1α protein was not produced in the HIF-1α knockout EPC cells. Apoptosis progression induced by camptothecin and viral hemorrhagic septicemia virus (VHSV) infection was severely inhibited by HIF-1α knockout, and the replication of VHSV was significantly retarded in HIF-1α knockout EPC cells. These results suggest that HIF-1α in EPC cells acts as a pro-apoptotic factor in the progression of apoptosis triggered by a DNA damaging agent and rhabdoviral infection.



This research was supported by Basic Science Research Program through the National Research Foundation (NRF) funded by the Ministry of Science, ICT and Future Planning of South Korea (NRF-2017R1C1B2003726).

Compliance with ethical standards

Conflict of interest

The authors declare no conflicts of interest.

Research involving human participants and/or animals

No part of this study was performed with human participants or animals by any of the authors.


  1. 1.
    Semenza GL (2007) Life with oxygen. Science 318:62–64CrossRefGoogle Scholar
  2. 2.
    Sitkovsky MV, Lukashev D, Apasov S, Kojima H, Koshiba M, Caldwell C, Ohta A, Thiel M (2004) Physiological control of immune response and inflammatory tissue damage by hypoxia-inducible factors and adenosine A2A receptors. Annu Rev Immunol 22:657–682CrossRefGoogle Scholar
  3. 3.
    Poellinger L, Johnson RS (2004) HIF-1 and hypoxic response: the plot thickens. Curr Opin Genet Dev 14:81–85CrossRefGoogle Scholar
  4. 4.
    Pugh CW, Ratcliffe PJ (2003) Regulation of angiogenesis by hypoxia: role of the HIF system. Nat Med 9:677–684CrossRefGoogle Scholar
  5. 5.
    Semenza GL (2008) O2 sensing: only skin deep? Cell 133:206–208CrossRefGoogle Scholar
  6. 6.
    Greijer AE, van der Wall E (2004) The role of hypoxia inducible factor 1 (HIF-1) in hypoxia induced apoptosis. J Clin Pathol 57:1009–1014CrossRefGoogle Scholar
  7. 7.
    Bruick RK (2000) Expression of the gene encoding the proapoptotic Nip3 protein is induced by hypoxia. Proc Natl Acad Sci USA 97:9082–9087CrossRefGoogle Scholar
  8. 8.
    Carmeliet P, Dor Y, Herbert JM, Fukumura D, Brusselmans K, Dewerchin M, Neeman M, Bono F, Abramovitch R, Maxwell P, Koch CJ, Ratcliffe P, Moons L, Jain RK, Collen D, Keshert E (1998) Role of HIF-1α in hypoxia-mediated apoptosis, cell proliferation and tumour angiogenesis. Nature 394:485–490CrossRefGoogle Scholar
  9. 9.
    Kilic M, Kasperczyk H, Fulda S, Debatin KM (2007) Role of hypoxia inducible factor-1α in modulation of apoptosis resistance. Oncogene 26:2027–2038CrossRefGoogle Scholar
  10. 10.
    Sasabe E, Tatemoto Y, Li D, Yamamoto T, Osaki T (2005) Mechanism of HIF-1α-dependent suppression of hypoxia-induced apoptosis in squamous cell carcinoma cells. Cancer Sci 96:394–402CrossRefGoogle Scholar
  11. 11.
    Yu EZ, Li YY, Liu XH, Kagan E, McCarron RM (2004) Antiapoptotic action of hypoxia-inducible factor-1α in human endothelial cells. Lab Investig 84:553–561CrossRefGoogle Scholar
  12. 12.
    Elks PM, van Eeden FJ, Dixon G et al (2011) Activation of hypoxia-inducible factor-1alpha (Hif-1α) delays inflammation resolution by reducing neutrophil apoptosis and reverse migration in a zebrafish inflammation model. Blood 118:712–722CrossRefGoogle Scholar
  13. 13.
    Jackson SA, McKenzie RE, Fagerlund RD, Kieper SN, Fineran PC, Brouns SJ (2017) CRISPR-Cas: adapting to change. Science 356:15056CrossRefGoogle Scholar
  14. 14.
    Chakrapani V, Patra SK, Panda RP, Rasal KD, Jayasankar P, Barman HK (2016) Establishing targeted carp TLR22 gene disruption via homologous recombination using CRISPR/Cas9. Dev Comp Immunol 61:242–247CrossRefGoogle Scholar
  15. 15.
    Chen J, Jiang D, Tan D, Fan Z, Wei Y, Li M, Wang D (2017) Heterozygous mutation of eEF1A1b resulted in spermatogenesis arrest and infertility in male tilapia, Oreochromis niloticus. Sci Rep 7:43733CrossRefGoogle Scholar
  16. 16.
    Homma N, Harada Y, Uchikawa T, Kamei Y, Fukamachi S (2017) Protanopia (red color-blindness) in medaka: a simple system for producing color-blind fish and testing their spectral sensitivity. BMC Genet 18:10CrossRefGoogle Scholar
  17. 17.
    Lau ES, Zhang Z, Qin M, Ge W (2016) Knockout of zebrafish ovarian aromatase gene (cyp19a1a) by TALEN and CRISPR/Cas9 leads to all-male offspring due to failed ovarian differentiation. Sci Rep 6:37357CrossRefGoogle Scholar
  18. 18.
    Gerri C, Marín-Juez R, Marass M, Marks A, Maischein HM, Stainier DYR (2017) Hif-1α regulates macrophage-endothelial interactions during blood vessel development in zebrafish. Nat Commun 8:15492CrossRefGoogle Scholar
  19. 19.
    Dehler CE, Boudinot P, Martin SA, Collet B (2016) Development of an efficient genome editing method by CRISPR/Cas9 in a fish cell line. Mar Biotechnol 18:449–452CrossRefGoogle Scholar
  20. 20.
    Liu LF, Desai SD, Li TK, Mao Y, Sun M, Sim SP (2000) Mechanism of action of camptothecin. Ann N Y Acad Sci 922:1–10CrossRefGoogle Scholar
  21. 21.
    Skall HF, Olesen NJ, Mellergaard S (2005) Viral haemorrhagic septicaemia virus in marine fish and its implications for fish farming—a review. J Fish Dis 28:509–529CrossRefGoogle Scholar
  22. 22.
    Ammayappan A, Vakharia VN (2011) Nonvirion protein of novirhabdovirus suppresses apoptosis at the early stage of virus infection. J Virol 85:8393–8402CrossRefGoogle Scholar
  23. 23.
    Eléouët JF, Druesne N, Chilmonczyk S, Monge D, Dorson M, Delmas B (2001) Comparative study of in-situ cell death induced by the viruses of viral haemorrhagic septicaemia (VHS) and infectious pancreatic necrosis (IPN) in rainbow trout. J Comp Pathol 124:300–307CrossRefGoogle Scholar
  24. 24.
    Emerling BM, Weinberg F, Liu JL, Mak TW, Chandel NS (2008) PTEN regulates p300-dependent hypoxia-inducible factor 1 transcriptional activity through Forkhead transcription factor 3a (FOXO3a). Proc Natl Acad Sci USA 105:2622–2627CrossRefGoogle Scholar
  25. 25.
    Rytkonen KT, Akbarzadeh A, Miandare HK, Kamei H, Duan C, Leder EH, Williams TA, Nikinmaa M (2013) Subfunctionalization of cyprinid hypoxia-inducible factors for roles in development and oxygen sensing. Evolution 67:873–882CrossRefGoogle Scholar
  26. 26.
    Rytkonen KT, Prokkola JM, Salonen V, Nikinmaa M (2014) Transcriptional divergence of the duplicated hypoxia-inducible factor alpha genes in zebrafish. Gene 541:60–66CrossRefGoogle Scholar
  27. 27.
    Kajimura S, Aida K, Duan C (2006) Understanding hypoxia-induced gene expression in early development: in vitro and in vivo analysis of hypoxia-inducible factor 1-regulated zebra fish insulin-like growth factor binding protein 1 gene expression. Mol Cell Biol 26:1142–1155CrossRefGoogle Scholar
  28. 28.
    Tausendschon M, Rehli M, Dehne N, Schmidl C, Doring C, Hansmann ML, Brune B (2015) Genome-wide identification of hypoxia-inducible factor-1 and -2 binding sites in hypoxic human macrophages alternatively activated by IL-10. Biochim Biophys Acta 1849:10–22CrossRefGoogle Scholar
  29. 29.
    Soitamo AJ, Rabergh CM, Gassmann M, Sistonen L, Nikinmaa M (2001) Characterization of a hypoxia-inducible factor (HIF-1α) from rainbow trout. Accumulation of protein occurs at normal venous oxygen tension. J Biol Chem 276:19699–19705CrossRefGoogle Scholar
  30. 30.
    Houghton PJ, Cheshire PJ, Hallman JD 2nd, Lutz L, Friedman HS, Danks MK, Houghton JA (1995) Efficacy of topoisomerase I inhibitors, topotecan and irinotecan, administered at low dose levels in protracted schedules to mice bearing xenografts of human tumors. Cancer Chemother Pharmacol 36:393–403CrossRefGoogle Scholar
  31. 31.
    Wen Y, Wang Y, Liu X, Zhang W, Xiong X, Han Z, Liang X (2017) Camptothecin-based nanodrug delivery systems. Cancer Biol Med 14:363–370CrossRefGoogle Scholar
  32. 32.
    Cramer T, Yamanishi Y, Clausen BE, Forster I, Pawlinski R, Mackman N, Haase VH, Jaenisch R, Corr M, Nizet V, Firestein GS, Gerber HP, Ferrara N, Johnson RS (2003) HIF-1α is essential for myeloid cell-mediated inflammation. Cell 112:645–657CrossRefGoogle Scholar
  33. 33.
    Werth N, Beerlage C, Rosenberger C, Yazdi AS, Edelmann M, Amr A, Bernhardt W, von Eif C, Becker K, Schafer A, Peschel A, Kempf VA (2010) Activation of hypoxia inducible factor 1 is a general phenomenon in infections with human pathogens. PloS One 5:e11576CrossRefGoogle Scholar
  34. 34.
    Kim MS, Lee JA, Lim KH (2016) Effects of a broad-spectrum caspase inhibitor, Z-VAD(OMe)-FMK, on viral hemorrhagic septicemia virus (VHSV) infection-mediated apoptosis and viral replication. Fish Shellfish Immunol 51:41–45CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Graduate School of Integrated BioindustrySejong UniversitySeoulSouth Korea
  2. 2.Department of Aquatic Life MedicinePukyong National UniversityBusanSouth Korea

Personalised recommendations