Effect Of FoxO1-shRNA Plasmid On Expression Of AQP9 In Human Normal Hepatic Cell Line

Objective To select the most potent plasmid vector using transient transfection of distinct shRNA plasmids specific for FoxO1 to human normal hepatic cell line L-02, and to evaluate the effect of FoxO1 gene silence on expression of AQP9 in L-02.Methods Four shRNA plasmids specific for FoxO1 were transfected to L-02 by liposome, and the transfection efficiencies were evaluated via fluorescence microscope. RT-PCR and western-blot were performed to select the most potent FoxO1-shRNA, which was next transfected to L-02 afresh to determine its effect on expression of AQP9 at transcriptional and translational levels. The time transcription for AQP9 regulated by FoxO1 was gained by real-time PCR in different time after transfaction.Results FoxO1-shRNA2 was the most potent vector among the four FoxO1-specific plasmids (FoxO1-shRNA1-4) which led to different gene silence effect. In contrast to control group, the mRNA and protein expression of FoxO1 were inhibited significantly by FoxO1-shRNA2 (P<0.05), and subsequently, the expression of AQP9 were inhibited at both transcriptional and translational levels (P<0.05). The exspression of AQP9 mRNA observed by real-time PCR decreased in time-depended, and the peaked at 60h after transfaction (P<0.05).Conclusions The expression of AQP9 in hepatic cells is decreased by FoxO1-specific plasmids, The expression of AQP9 in hepatic cells is decreased by FoxO1-specific plasmids. The determination of AQP9 transcription time by real-time PCRh provide experiment support in researching the dynamic variation of the combination between FoxO1 and IRE within AQP9 promoter by Chromatin Immunoprecipitation.