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

Abstract

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.

FIGURE 1.

VEGF expression in human cells treated with siRNA targeting the endogenous VEGF promoter. (A) VEGF expression in HeLa and ARPE19 cells, transfected with siRNAs designed to specific sites within the endogenous VEGF gene promoter. (B) VEGF gene expression in HeLa cells transfected with siVFp(−992) and its variants. VEGF expression from cells transfected with each siRNA is normalized to “Mock” transfected cells and presented as the percentage mean ± SD from three independent experiments, with three replicate samples per experiment; (*) p < 0.05.

FIGURE 2.

Luciferase assay performed in human cells co-transfected with VEGF promoter containing reporter plasmid and siRNA targeting the VEGF promoter. (A) Schematic representation of the luciferase reporter plasmids engineered to contain either wild-type (pVEGFprom2.3-Luc), mutated (pVEGFprom2.3.VFp992mut-Luc), or deleted (pVEGFprom2.3.VFp992del-Luc) promoter target sequences. Mismatched bases are underlined, and deleted bases are indicated with a hyphen. (B) Luciferase (firefly) gene activity from HeLa cells transfected with each reporter plasmid when treated with siVFp(−992) or its variants. Luciferase activity is normalized to that from cells transfected with vector without siRNA (Vector alone) and is presented as the percentage mean ± SD from three independent experiments, with three replicate samples per experiment; (*) p < 0.05.

FIGURE 3.

Luciferase assay performed in human cells containing an integrated HIV_SF2-LTR promoter driven reporter gene and co-transfected with siRNA targeting the HIV_SF2-LTR promoter and Tat expression plasmid, pSV40.Tat-86. (A) Schematic representation of the luciferase reporter plasmids engineered to contain either wild-type (pGL4.20.SF2-LTR-Luc) or mutated (pGL4.20.SF2mut-LTR-Luc) promoter target sequences within the HIV_SF2-LTR promoter. Mismatched bases are underlined. (B) Luciferase (firefly) gene activity from HeLa cells containing an integrated reporter gene when treated with siSF2-247 or its variants. Tat-induced firefly luciferase activity is normalized to that from cells not transfected with siRNA (“Mock”) and is presented as the percentage mean ± SD from three independent experiments, with three replicate samples per experiment; (*) p < 0.05.

FIGURE 4.

Protein expression analysis from human cells transfected with siRNA complementary to the VEGF gene promoter or GRB2 mRNA. (A) VEGF expression in HeLa cells transfected with siGRB2 or control siRNA. VEGF expression from cells transfected with each siRNA is normalized to “Mock” transfected cells and presented as the percentage mean ± SD from three independent experiments, with three replicate samples per experiment; (*) p < 0.05. (B) Western blot analysis for the detection and quantitation of GRB2 protein extracted from HeLa cells treated with siVFp(−992) or control siRNA. A representative blot is shown with samples loaded per lane, as follows: (lane 1) Mock transfected cells; (lane 2) siVFp(−992); (lane 3) siVFp(−992)m1; and (lane 4) siGRB2. The intensity of each band for GRB2 was quantified by densitometry and normalized to that for β-actin. GRB2 protein levels from HeLa cells transfected with each siRNA are shown as a ratio relative to Mock transfected cells and presented as the percentage mean ± range from two independent experiments, with two replicate samples per experiment.