[Construction of shRNA eucharyotic expression plasmid targeted human VIGILIN and the investigation of VIGILIN's effect on hepatocarcinoma cell cycle].

OBJECTIVE: To construct a shRNA eucharyotic expression plasmid against human VIGILIN and explore possible relation between human VIGILIN and HepG2 cell cycle.

METHODS: We constructed the shRNA eucharyotic expression plasmid targeted human VIGILIN, and transfected HepG2 cells with shRNA expression plasmid pSIREN-VIG, then determined the expression of VIGILIN mRNA and protein in HepG2 cells by RT-PCR and Western-blot, analysed alteration of cell cycle using FACS.

RESULTS: The plasmid pSIREN-VIG can effectively and specifically inhibit the expression of human VIGILIN. After transfection 48 hours, the expression of VIGILIN was significantly decreased. Due to knockdown of human VIGILIN, cell cycle is impaired and cells are arrested in G2/M phase. The proportion of G2/M phase of all groups were listed as: C group (untreated wild HepG2 cells) 2.4%, M group (HepG2 cells treated with transfection reagent) 4.9%, G group (HepG2 cells transfected with pSIREN-GFP) 6.5% and V group (HepG2 cells transfected with pSIREN-VIG) 9.4%.

CONCLUSION: We have successfully constructed a shRNA expression plasmid which could effectively and specifically inhibit the expression of human VIGILIN.