Inhibitory effects of lentivirus mediated RNA interference targeting human AQP1 gene on the proliferation of human colon carcinoma SW480 cells and the expression of VEGF

Purpose: The objective of the present study was to construct the lentiviral expression vector for RNA interference (RNAi) of human AQPl gene in colon carcinoma SW480 cells, which may provide research foundation for investigating the mechanisms that AQP1 gene functions for the proliferation of human colon carcinoma SW480 cells and the expression of VEGF in the cells. Methods: Four effective sequences of RNAi targeting human AQPl gene were confirmed. Both sense and antisense Oligo DNA of the targeting sequences were designed, synthesized and cloned into the lentiviral vector pEGFP-N1-3FLAG. After transfected with lentiviral vector of T293 cells, and the titer of the lentivirus was tested. SW480 cells were infected with the lentivirus and the expression of AQPl mRNA and protein in SW480 cells was detected by real-time quantitative polymerase chain reaction (RT-PCR) and Western blotting. The propagation of SW480 cells was detected by MTT and expression of VEGF in SW480 cells was detected by RT-PCR. Results: A recombinant lentiviral vector expressing shRNA against AQP1 gene was obtained confirmed by DNA sequencing. The titre of virus was 2x10(9) TU/ml. AQP1 mRNA and protein expression in SW480 cells after infected with lentiviral vector was decreased remarkably. The mRNA expression was 60%, respectively, compared with the control group. And the lentiviral shRNA expression vector reduced cell pro1 if eration 5 d after stable AQP1 gene knock-down in SW480 cells. Meanwhile, the expression of VEGF mRNA was inhibited. Discussion: The lentiviral shRNA expression vector targeting human AQP1 gene capable of stable AQP1 gene knock-down SW480 cells has been successfully constructed, which provides a basis for further study of mechanisms that AQP1 gene acts for proliferation of colon carcinoma. And preliminary conclution was drawn that AQP1 gene may promote the growth of SW480 cells and the expression of VEGF.