Tumor Biology

, Volume 37, Issue 5, pp 5847–5855 | Cite as

The construction and proliferative effects of a lentiviral vector capable of stably overexpressing SPINK1 gene in human pancreatic cancer AsPC-1 cell line

  • Jing Zhang
  • Dongmei Wang
  • Na Hu
  • Qian Wang
  • Shanice Yu
  • Jun Wang
Original Article

Abstract

This study aims to design and generate recombinant lentiviral vector containing the complete coding sequence (CDS) region of human serine protease inhibitor Kazal type 1 gene (SPINK1) and establish a human pancreatic cancer cell line (AsPC-1) stably overexpressing SPINK1. Then, to assess the proliferative and oncogenic effects of upregulated SPINK1 gene on pancreatic cancer AsPC-1 cells using different methods. Initially, the target coding sequence (CDS) of SPINK1 was amplified by polymerase chain reaction (PCR) and the synthesized product was subsequently subcloned into the lentiviral vector. The construction of recombinant SPINK1 gene was verified by the restriction digestion and sequencing analysis. The lentiviral particles carrying SPINK1 gene were produced by co-transfection of the recombination lentiviral vector and the packaging mix plasmids into 293 T cells and filtered and concentrated before AsPC-1 cells were infected by the virus particles. The cells transduced were differentially selected with puromycin, and the clones that highly expressed SPINK1 were chosen by real-time PCR and confirmed by Western blot after 7 weeks. The stably transduced AsPC-1 cell line showed significantly increased metabolic and proliferative capability using CCK-8 and Trypan Blue assays (P < 0.001). Cell cycle and DNA content analysis by flow cytometry showed that upregulated SPINK1 elicited significant increase in the percentage of AsPC-1 cells in the S and G2/M phase (P < 0.001). Clone formation assay demonstrated that the number of the colonies formed in the experimental group was greater than that in the control parental cells (P < 0.001). It was concluded that a stable AsPC-1 cell line capable of overexpressing SPINK1 had been successfully created, and that the proliferative capacity of AsPC-1 pancreatic cancer cells was significantly raised by SPINK1 upregulation as well as the ability of a single AsPC-1 cell to grow into a colony.

Keywords

Serine protease inhibitor Kazal type 1 (SPINK1) Lentiviral vector Pancreatic cancer Proliferation 

Notes

Acknowledgments

This study was financially supported by the National Natural Science Foundation of China (NSFC), Grant no.: 81270542, and Research Foundation of Dalian Medical University.

Compliance with ethical standards

Conflicts of interest

None

Supplementary material

13277_2015_4405_Fig6_ESM.jpg (2.5 mb)
Supplementary Figure 1

The sequencing data for the constructed recombinant lentiviral vector. The whole SPINK1 coding sequence subcloned in pLenti-CMV-2A-GFP vector was confirmed by the sequencing. The inserted EcoRI (site), “A” nucleotide, Kozak sequence, CDS of SPINK1 gene, and BamHI (site) were represented in the sequence graph. (JPG 2511 kb)

13277_2015_4405_Fig7_ESM.jpg (1.5 mb)
Supplementary Figure 2

The amplification plot and melting curve from q-PCR reaction for the detection of mRNA expression of SPINK1. a An amplification plot showed the variation of log (ΔRn) with PCR cycle number. The results revealed three typical “S-shaped” amplification curves. b An amplicon either from 293 T (positive control), stable clone #10 or parental AsPC-1 cells produced only one peak in the plot. (JPG 1575 kb)

References

  1. 1.
    Yutong H et al. Pancreatic cancer incidence and mortality patterns in China, 2011. Chin J Cancer Res. 2015;27(1):29–37.Google Scholar
  2. 2.
    Kozak G, Blanco FF, Brody JR. Novel targets in pancreatic cancer research. Semin Oncol. 2015;42(1):177–87.CrossRefPubMedGoogle Scholar
  3. 3.
    Ida S et al. SPINK1 status in colorectal cancer, impact on proliferation, and role in colitis-associated cancer. Mol Cancer Res. 2015;13(7):1130–8.CrossRefPubMedGoogle Scholar
  4. 4.
    Ogata N. Demonstration of pancreatic secretory trypsin inhibitor in serum-free culture medium conditioned by the human pancreatic carcinoma cell line CAPAN-1. J Biol Chem. 1988;263(26):13427–31.PubMedGoogle Scholar
  5. 5.
    Nobuyuki O et al. Serine protease inhibitor Kazal type 1 and epidermal growth factor receptor are expressed in pancreatic tubular adenocarcinoma, intraductal papillary mucinous neoplasm, and pancreatic intraepithelial neoplasia. J Hepatobiliary Pancreat Sci. 2013;20(6):620–7.CrossRefGoogle Scholar
  6. 6.
    Marchbank T, Mahmood A, Playford RJ. Pancreatic secretory. Trypsin inhibitor causes autocrine-mediated migration and invasion in bladder cancer and phosphorylates the EGF receptor, Akt2 and Akt3, and ERK1 and ERK2. Am J Physiol Renal Physiol. 2013;305(3):382–9.CrossRefGoogle Scholar
  7. 7.
    Ohmachi Y et al. Overexpression of pancreatic secretory trypsin inhibitor in pancreatic cancer. Evaluation of its biological function as a growth factor. Int J Pancreatol. 1994;15(1):65–73.PubMedGoogle Scholar
  8. 8.
    Ohmuraya M, Yamamura K. The roles of serine protease inhibitor Kazal type 1 (SPINK1) in pancreatic diseases. J Exp Anim. 2011;60(5):433–44.CrossRefGoogle Scholar
  9. 9.
    Ozaki N et al. Serine protease inhibitor Kazal type 1 promotes proliferation of pancreatic cancer cells through the epidermal growth factor receptor. Mol Cancer Res. 2009;7(9):1572–81.CrossRefPubMedGoogle Scholar
  10. 10.
    López-Sáez JF et al. Cell proliferation and cancer. Histol Histopathol. 1998;13(14):1197–214.PubMedGoogle Scholar
  11. 11.
    Croce CM. Oncogenes and cancer. N Engl J Med. 2008;358:502–11.CrossRefPubMedGoogle Scholar
  12. 12.
    Taylor HE et al. The innate immune factor apolipoprotein L1 restricts HIV-1 infection. J Virol. 2014;1:592–603.CrossRefGoogle Scholar
  13. 13.
    Shearer RF, Saunders DN. Experimental design for stable genetic manipulation in mammalian cell lines: lentivirus and alternatives. Genes Cells. 2015;20(1):1–10.CrossRefPubMedGoogle Scholar
  14. 14.
    Chen Y et al. Construction and application of a lentiviral vector of single-chain variable fragment antibody against human hepatocyte growth factor receptor. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2014;30(9):960–3. Chinese.PubMedGoogle Scholar
  15. 15.
    Appaiahgari MB, Vrati S. Adenoviruses as gene/vaccine delivery vectors: promises and pitfalls. Expert Opin Biol Ther. 2015;15(3):337–51.CrossRefPubMedGoogle Scholar
  16. 16.
    Tominaga H et al. A water-soluble tetrazolium salt useful for colorimetric cell viability assay. Anal Commun. 1999;36:47–50.CrossRefGoogle Scholar
  17. 17.
    Spyratos F et al. Biology of the cell DNA content and cell cycle analysis by flow cytometry in clinical samples: application in breast cancer. Biol Cell. 1993;78(1–2):69–72.CrossRefPubMedGoogle Scholar
  18. 18.
    Favoni RE, de Cupis A. The role of polypeptide growth factors in human carcinomas: new targets for a novel pharmacological approach. Pharmacol Rev. 2000;52(2):179–206.PubMedGoogle Scholar
  19. 19.
    Wood CE et al. Scientific and regulatory policy committee (SRPC) review: interpretation and use of cell proliferation data in cancer risk assessment. Toxicol Pathol. 2015;43(6):760–75.CrossRefPubMedGoogle Scholar
  20. 20.
    Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144(5):646–74.CrossRefPubMedGoogle Scholar
  21. 21.
    Niinobu T et al. Identification and characterization of receptors specific for human pancreatic secretory trypsin inhibitor. J Exp Med. 1990;172:1133–42.CrossRefPubMedGoogle Scholar
  22. 22.
    Ohmuraya M, Yamamura K. Roles of serine protease inhibitor Kazal type 1 (SPINK1) in pancreatic diseases. Exp Anim. 2011;60(5):433–44.CrossRefPubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2015

Authors and Affiliations

  • Jing Zhang
    • 1
  • Dongmei Wang
    • 2
  • Na Hu
    • 1
  • Qian Wang
    • 3
  • Shanice Yu
    • 3
  • Jun Wang
    • 1
  1. 1.Department of Pathophysiology, College of Basic Medical SciencesDalian Medical UniversityDalianChina
  2. 2.Department of Experimental Functionality, College of Basic Medical SciencesDalian Medical UniversityDalianChina
  3. 3.Nanjing Applied Biological Materials Inc.NanjingChina

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