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 Westemblotting 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 calls 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× 10^9TU/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.
Jianxin Jiang Ming Shen Renyi Qin Rui Tian Jing Li Min Wang
