Abstract
Objective
The aim of the study was to investigate the interference and anti-tumor effects of lentiviral vector of miRNA targeting IGF1R gene regulated by survivin promoter.
Methods
The fragment of the survivin promoter was acquired by PCR amplification and inserted into pPRIME to recombinant plasmid sur-pPRIME. The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was designed, synthesized and cloned into the sur-pPRIME vector, named sur-pPRIME-IGF1R-miR30-shRNA. Viruses were propagated on 293T cells. Viruses were purified by CsCI gradient according to standard techniques, and functional PFU titers were determined by plaque assay on 293 cells. The effect of sur-pPRIME-IGF1R-miR30-shRNA on IGF1R expression of Hep3B cells was detected by RT-PCR and Western blot. The antitumor potential of sur-pPRIME-IGF1R-miR30-shRNA to Hep3B cells was evaluated by CCK-8 assay.
Results
sur-pPRIME-IGF1R-miR30-shRNA was constructed successfully. Functional PFU titers of sur-pPRIME-IGF1R-miR30-shRNA were 4.58×109 PFU/mL. Sur-pPRIME-IGF1R-miR30-shRNA was more effective to inhibit IGF1R expression in mRNA or protein levels and the proliferation of Hep3B cells.
Conclusion
sur-pPRIME-IGF1R-miR30-shRNA expressing IGF1R-siRNA can inhibit IGF1R expression and may be used for further investigation of gene therapy of liver cancer.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Fang L, Pu YY, Hu XC, et al. Antiangiogenesis gene armed tumor-targeting adenovirus yields multiple antitumor activities in human HCC xenografts in nude mice. Hepatol Res, 2010, 40: 216–228.
Konno R, Yamakawa H, Utsunomiya H, et al. Expression of survivin and Bcl-2 in the normal human endometrium. Mol Hum Reprod, 2000, 6: 529–534.
Jeha S, Luo XN, Beran M, et al. Antisense RNA inhibition of phosphoprotein p18 expression abrogates the transformed phenotype of leukemic cells. Cancer Res, 1996, 56: 1445–1450.
Su CQ, Na ML, Chen J, et al. Gene-viral cancer therapy using dualregulated oncolytic adenovirus with antiangiogenesis gene for increased efficacy. Mol Cancer Res, 2008, 6: 568–575.
Lange SS, Takata K, Wood RD. DNA polymerases and cancer. Nat Rev Cancer, 2011, 11: 96–110.
Lois C, Hong EJ, Pease S, et al. Germline transmission and tissuespecific expression of transgenes delivered by lentiviral vectors. Science, 2002, 295: 868–872.
Pfeifer A, Ikawa M, Dayn Y, et al. Transegenesis by lentiviral vectors: lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos. Proc Natl Acad Sci USA, 2002, 99: 2140–2145.
Lai Z, Brady RO. Gene transfer into the central nervous system in vivo using a recombinant lentivirus vector. J Neurosci Res, 2002, 67: 363–371.
Zufferey R, Dull T, Mandel RJ, et al. Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J Virol, 1998, 72: 9873–9880.
Yu X, Zhan X, D’Costa J, et al. Lentiviral vectrs with two independent internal promoters transfer high-level expression of multiple transgenes to human hematopoietic stem-progenitor cells. Mol Ther, 2003, 7: 827–838.
Zhao J, Pettigrew GJ, Thomas J, et al. Lentiviral vectors for delivery of genes into neonatal and adult ventricular cardiac myocytes in vitro and in vivo. Basic Res Cardiol, 2002, 97: 348–358.
Bauer G, Dao MA, Case SS, et al. In vivo biosafety model to assess the risk of adverse events from retroviral and lentiviral vectors. Mol Ther, 2008, 16: 1308–1315.
Baba K, Goto-Koshino Y, Mizukoshi F, et al. Inhibition of the replication of feline immunodeficiency virus by lentiviral vector-mediated RNA interference in feline cell lines. J Vet Med Sci, 2008, 70: 777–783.
Zeng Y. Principles of micro-RNA production and maturation. Oncogene, 2006, 25: 6156–6162.
Navarro M, Baserga R. Limited redundancy of survival singals from the type 1 insulin-like growth factors receptor. Endocrinology, 2001, 142: 1073–1081.
Author information
Authors and Affiliations
Corresponding author
Additional information
Supported by the grants from the Education Departmental Natural Science Research Funds of Jiangsu Provincial Higher School of China (No. 09KJB320018, No. 09KJB310016).
Rights and permissions
About this article
Cite this article
Niu, J., Liu, Z., Song, Y. et al. Research on construction and identification of lentiviral vector of expressing miRNA targeting IGF1R gene regulated by survivin promoter and its inhibition to liver cancer cell growth. Chin. -Ger. J. Clin. Oncol. 10, 705–710 (2011). https://doi.org/10.1007/s10330-011-0867-7
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10330-011-0867-7