Abstract
Objective
To construct a eukaryotic expression vector for RNA interference of the human cyclinD1 gene, and to detect its interference effect in human ovarian cancer cells (HO-8910).
Methods
Four target gene segments were synthesized and cloned into the pSUPER vector respectively to construct four recombinant eukaryotic expression vectors, pSUPER-C1∼4. The four recombinant vectors were identified by enzyme digestion analysis and DNA sequencing. Then HO-8910 cells were transfected with the pSUPER-C1∼4 vectors and subjected to G418 selection. In G418-resistant cells, the interference effect was detected by RT-PCR.
Results
Enzyme digestion analysis and DNA sequencing showed that the target segments were cloned into the pSUPER vector. The four recombinant vectors inhibited transcription of the cyclinD1 gene. The pSUPER-C2 vector had a better interference effect.
Conclusion
The sequence-specific siRNA effectively interfered with expression of the cyclinD1 gene that was selected. The transcription and expression of the cyclinD1 gene were inhibited effectively by the constructed RNAi eukaryotic expression vectors in the ovarian cancer cells. These results indicate that it is possible to search for a new tumor gene therapy method.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Dimova I, Zaharieva B, Raicheva S, et al. Association of cyclinD1 copy number changes with histological type in ovarian tumors. Acta Oncol 2004; 43: 675–679.
Krichevsky AM, Kosik KS. RNAi functions in cultured mammalian neurons. Proc Natl Acad Sci USA 2002; 99: 11926–11929.
Peek AS, Behlke MA. Design of active small interfering RNAs. Curr Opin Mol Ther 2007; 9: 110–118.
Ambion. Five ways to produce si RNA[J/OL]. http://www.ambion.com/techlib/tn/103/2.html.2005,12.
Wassenegger M. The role of the RNAi machinery in heterochromatin formation. Cell 2005; 122: 13–16.
Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells. Science 2002; 296: 550–553.
Hannon GJ. RNA interference. Nature 2002; 418: 244–251.
Mello CC, Conte D Jr. Revealing the world of RNA interference. Nature 2004; 431: 338–342.
Natt F. siRNAs in drug discovery: target validation and beyond. Curr Opin Mol Ther 2007; 9: 242–247.
de Fougerolles A, Vornlocher HP, Maraganore J, et al. Interfering with disease: a progress report on siRNA-based therapeutics. Nat Rev Drug Discov 2007; 6: 443–453.
Tashiro E, Tsuchiya A, Imoto M. Functions of cyclin D1 as an oncogene and regulation of cyclin D1 expression. Cancer Sci 2007; 98: 629–635.
Li Z, Wang C, Prendergast GC, et al. Cyclin D1 functions in cell migration. Cell Cycle 2006; 5: 2440–2442.
Shankland SJ, Wolf G. Cell cycle regulatory proteins in renal disease: role in hypertrophy, proliferation, and apoptosis. Am J Physiol Renal Physiol 2000; 278: 515–529.
Reddy HK, Mettus RV, Rane SG, et al. Cyclin-dependent kinase 4 expression is essential for neu-induced breast tumorigenesis. Cancer Res 2005; 65: 10174–10178.
Yan KX, Liu BC, Shi XL, et al. Role of cyclinD1 and CDK4 in the carcinogenesis induced by silica. Biomed Environ Sci 2005; 18: 286–296.
Caldon CE, Daly RJ, Sutherland RL, et al. Cell cycle control in breast cancer cells. J Cell Biochem 2006; 97: 261–274.
Author information
Authors and Affiliations
Corresponding author
About this article
Cite this article
Yu, D., Hao, L., Li, Y. et al. Construction and identification of a vector expressing RNA interference aimed at the human cyclinD1 gene and its expression in vitro. Chin. J. Clin. Oncol. 4, 338–342 (2007). https://doi.org/10.1007/s11805-007-0338-7
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/s11805-007-0338-7