Inhibitory effects of lentivirus mediated RNA interference targeting human Wip1 gene on the prolif-eration of human glioma U251 cells

Abstract

Objective To construct the lentiviral expression vector for RNA interference (RNAi) of Wipl gene in glioma cells and provide research foundation for studying the mechanisms that wild-type p53-induced phosphatase 1 (Wip1 ) gene functions for malignant proliferation of gliomas. Methods Three effective sequences of RNAi targeting Wip1 were confirmed. Both sense and antisense Oligo DNA of the tar-geting sequence were designed, synthesized and cloned into the pFU-GW-iRNA vector. After cotransfectsd with lentiviral vector of T293 cells, the lentivirus was produced and the titer of virus was tested. U251 cells were infected with the lentivirus and the expression of Wip1 mRNA and protein in U251 cells was detected by real-time quantitative polymerase chain reaction (PCR) and Western blotting. The propagation of U251 was detected by CCK-8 and expression of bcl-2 was detected by Western blotting. Results A recombinant lentiviral vector expressing shRNAs against Wip1 gene was obtained and confirmed by DNA sequencing. The titer of virus was 3×10~8-8×10~8 TU/ml. Wipl mRNA and protein expression in U251 cells after infec-ted with lentiviral vector was decreased remarkably. The mRNA expression was 36. 3% ,32. 9% and 23.8% respectively, compared with the control group. And the lentiviral shRNA expression vector reduced cell pro-liferation by 35. 1% 4 d after stable Wipl gene knock-down in U251 cells. Conclusion The lentiviral shRNA expression vector targeting human Wipl gene capable of stable Wipl gene knock-down in U251 cells has been successfully constructed,which provides a basis for further study of mechanisms that Wip1 gene acta for malignant proliferation of gliomas. And preliminary conclution was drawn that Wipl gene may promote the growth of U251 cells. Key words: Wip1 gene; Glioma; RNA interference; Lentiviral