Intended transcriptional silencing with siRNA results in gene repression through sequence-specific off-targeting

Transcriptional gene silencing has been reported with siRNA targeting the promoter region of genes. We tested several siRNAs directed against the human VEGF promoter. Of these, siVFp(-992) exhibited > or =50% suppression of VEGF production in two human cell lines. To determine the specificity of this siRNA-mediated suppression, plasmids were prepared to express a luciferase reporter under the control of VEGF promoters featuring wild-type, mutated, or deleted target sequences. siRNA transfection assays established sequence-specific inhibition of luciferase from the reporter plasmid featuring the wild-type VEGF promoter. However, siVFp(-992) also suppressed the luciferase expression from the plasmids with mutated or deleted target sites, suggesting that silencing was due to a sequence-specific off-target phenomenon, and this was supported by subsequent microarray and bioinformatics analyses. To determine if our concerns regarding the specificity of promoter targeting siRNAs were relevant to other systems where RNA-mediated transcriptional silencing had been previously reported, we tested a published small RNA sequence directed to the HIV(SF2)-LTR promoter. siRNA transfection assays performed in human cells expressing a luciferase reporter gene under the control of the HIV(SF2)-LTR promoter revealed significant suppression whether the target sequence was intact or mutated, or when the entire HIV(SF2)-LTR was replaced by an irrelevant promoter. These data stress the need to examine target specificity when conducting investigations into transcriptional gene regulation with siRNA.