RESUMO
Objective To study the effects of inhibiting forkhead transcription factor O1 (FoxO1)expression by small interference RNA (siRNA) on the glucose utilization of insulin resistant HepG-2 cell line and the mechanism.Methods FoxO1 gene-targeted siRNA vector,which carried a red fluorescence,was constructed and then confirmed by DNA sequencing.HepG-2 cells were induced to a status of insulin resistance by being exposed to 10-6 mol/L insulin for 24 h.The study included 4 groups: HepG-2 cell group cultured with normal medium (group A) ; insulin resistant HepG-2 cell group (group B) ; insulin resistant HepG-2 cell group into which FoxO1 siRNA vector was transfected (group C) ; insulin resistant HepG-2 cell group into which Lipofectamine2000 was added (group D).The transfection efficiency could be estimated by observing the expression of red fluorescence.The expression of FoxO1 mRNA was analyzed by RT-PCR.The expressions of IRS-2 and tyrosine phosphorylation were detected by Western blot and immunoprecipitation.Results FoxO1 gene-targeted siRNA vector was built successfully.The expression of red fluorescence was the strongest at 48 h after transfection.Compared with group A,the glucose consumption and the expression of IRS-2 tyrosine phosphorylation of group B were decreased (P<0.01),and expression of FoxO1 mRNA was increased (P<0.05) in group B.There was no difference in the expressions of IRS-2 protein between A and B groups.Compared with group B,the expression of FoxOl mRNA was decreased (P < 0.01),and the glucose consumption and expression of IRS-2 tyrosine phosphorylation were significantly increased (P<0.05) in group C.There was no difference between group D and group B in glucose consumption, FoxO1 mRNA, IRS-2 protein and IRS-2 tyrosine phosphorylation expressions.Conclusions Inhibiting the expression of FoxO1 gene in insulin resistant HepG-2 cells seems to improve insulin sensitivity by feedback regulating the IRS-2 tyrosine phosphorylation expressions.