Sirna On The Inhibition Of Wt1 Gene Expression

Objective To explore the feasibility of gene thearapy targeting on WT1 gene in leukemia, inhibitory effect of siRNA on WT1 gene expression was studied.Methods Three siRNA for WT1 were synthesized chemically and transfected into k562 cells and HEK293 cells . WT1 mRNA expression were detected by FQ-PCR. WT1 protein expression were detected by Western blot.Result The efficiency of liposomal reagent was low for suspended k562 cells.②The inhibitory effects were different for siRNAs designed different locations of wt1 gene. Si-wtl-1 was the best (P<0.05), But si-wt1-2 and si-wt1-3 had no inhibitory effect on WT1 mRNA expresion (P>0.05) .③There was no dose-dependent inhibitory effect for si-wtl-1 by the statistics analysis.④WT1 mRNA expression was reduced to (42.5±1.0)%and (25.3 + 1.5)% of the control at 24h and 48h transfected with 100nmol/L si-wtl-1 respectively (P<0.05) ,but restored to normal level at 72h.⑤Inhibitory effect of WT1 protein was evident with transfection of si-GAPDH after 72 hours by western blot .The inhibitory effect of si-wt1-1 was obvious and the effect is best at 96h especially (P<0.05), But si-wtl-2 and si-wtl-3 had no effect on WT1 protein expression by Western blot analysis (P>0.05 ) .Conclusions (1)There was related intimately between inhibitory effect of siRNA and designed location on wt1 gene. Inhibitory effect of si-wt1-1 was best the effect of transcription level exceeded 75%.(2)There is no obvious relation with the dose of siRNA between 60nmol/L to 120nmol/L.(3)The effective time were limited for siRNA synthesized chemically . The inhibitory effect was best at 48h and the effect is disappeared at 72h.