Abstract
Objective
To construct a lentiviral expression vector for RNA interference (RNAi) of human VIM gene; and assess its gene silencing effect in pancreatic cancer cell line Panc-1.
Methods
Three pairs of human VIM gene short hairpin RNA (shRNA) sequences were designed using a software available on-line and one pair came from document. After synthesis and annealing, four double-stranded oligonucleotides (dsOligo) were cloned into the pGCL-GFP/U6 plasmid, which were subsequently confirmed by polymerase chain reaction (PCR) and DNA sequencing analysis. Real-time PCR and Western-blotting were used to screen the effective pGCL-GFP-shRNA plasmid in 293T cells, then the most effective one was packed into the recombinant lentivirus Lv-VIM-shRNA with lentiviral packing materials pHelper 1.0 and pHelper 2.0 in 293T cells. The titer of lentivirus was determined by hole-by-dilution titer assay. The silencing effect of Lv-VIM-shRNA in Panc-1 cells were validated by real-time PCR and Western-blotting.
Results
An effective Lv-VIM-shRNA was successfully constructed. The titer of lentivirus was determined on 2 × 109 TU/mL. The expressions of VIM mRNA and vimentin were down-regluated in the Panc-1 cells infected with Lv-VIM-shRNA.
Conclusion
An effective Lv-VIM-shRNA could inhibit the expression of VIM gene in Panc-1 cells in vitro, which provides a tool for investigating the role of VIM gene in the signaling pathway involved in tumorigenesis and progression of pancreatic cancer and searching new therapeutic targets.
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References
Guo X, Cui Z. Current diagnosis and treatment of pancreatic cancer in China. Pancreas, 2005, 31: 13–22.
Bardeesy N, DePinho RA. Pancreatic cancer biology and genetics. Nat Rev Cancer, 2002, 2: 897–909.
Chen L, Liu Q, Qin R, et al. Amplification and functional characterization of MUC1 promoter and gene-virotherapy via a targeting adenoviral vector expressing hSSTR2 gene in MUC1-positive Panc-1 pancreatic cancer cells in vitro. Int J Mol Med, 2005, 15: 617–626.
Du ZY, Qin RY, Xia W, et al. Gene transfer of somatostatin receptor type 2 by intratumoral injection inhibits established pancreatic carcinoma xenografts. World J Gastroenterol, 2005, 11: 516–520.
Kumar M, Liu ZR, Thapa L, et al. Anti-angiogenic effects of somatostatin receptor subtype 2 on human pancreatic cancer xenografts. Carcinogenesis, 2004, 25: 2075–2081.
Feng Y, Huang T, Gao J, et al. Inhibition of metastatic progression of SSTR2 gene transfection mediated by adenovirus in human pancreatic carcinoma cells. J Huazhong Univ Sci Technolog Med Sci, 2006, 26: 68–71.
McInroy L, Määttä A. Down-regulation of vimentin expression inhibits carcinoma cell migration and adhesion. Biochem Biophys Res Commun, 2007, 360: 109–114.
Wang N, Stamenovic D. Mechanics of vimentin intermediate filaments. J Muscle Res Cell Motil, 2002, 23: 535–540.
Thiery JP, Sleeman JP. Complex networks orchestrate epithelial-mesenchymal transitions. Nat Rev Mol Cell Biol, 2006, 7: 131–142.
Paccione RJ, Miyazaki H, Patel V, et al. Keratin down-regulation in vimentin-positive cancer cells is reversible by vimentin RNA interference, which inhibits growth and motility. Mol Cancer Ther, 2008, 7: 2894–2903.
Wei J, Xu G, Wu M, et al. Overexpression of vimentin contributes to prostate cancer invasion and metastasis via src regulation. Anticancer Res, 2008, 28: 327–334.
Nakajima S, Doi R, Toyoda E, et al. N-cadherin expression and epithelial-mesenchymal transition in pancreatic carcinoma. Clin Cancer Res, 2004, 10: 4125–4133.
Shah AN, Summy JM, Zhang J, et al. Development and characterization of gemcitabine-resistant pancreatic tumor cells. Ann Surg Oncol, 2007, 14: 3629–3637.
Ohrt T, Schwille P. siRNA modifications and sub-cellular localization: a question of intracellular transport? Curr Pharm Des, 2008, 14: 3674–3685.
Blesch A. Lentiviral and MLV based retroviral vectors for ex vivo and in vivo gene transfer. Methods, 2004, 33: 164–172.
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Supported by a grant from the National Natural Science Foundation of China (No. 30600592).
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Jiang, J., Shen, M., Qin, R. et al. Construction of a lentiviral vector for RNA interference of human VIM gene and its silencing effect in pancreatic cancer cells. Chin. -Ger. J. Clin. Oncol. 8, 145–149 (2009). https://doi.org/10.1007/s10330-009-0017-7
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DOI: https://doi.org/10.1007/s10330-009-0017-7