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Knockdown of TNFR1 by the sense strand of an ICAM-1 siRNA: dissection of an off-target effect.

Clark PR, Pober JS, Kluger MS - Nucleic Acids Res. (2007)

Bottom Line: An EGFP construct incorporating the 3'UTR of TNFR1 was silenced by 736 siRNA and this effect was lost by mutagenesis of this complementary sequence.We show that this off-target effect is mediated by siRNA knockdown of TNFR1 via its sense strand.This may be the first example in which the off-target effect of an siRNA is actually responsible for the anticipated effect by acting to reduce expression of a protein (TNFR1) that normally regulates expression of the intended target (ICAM-1).

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Yale University School of Medicine, New Haven, CT 06510, USA. paul.clark@yale.edu

ABSTRACT
Tumor necrosis factor (TNF) initiates local inflammation by triggering endothelial cells (EC) to express adhesion molecules for leukocytes such as intercellular adhesion molecule-1 (ICAM-1 or CD54). A prior study identified siRNA molecules that reduce ICAM-1 expression in cultured human umbilical vein EC (HUVEC). One of these, ISIS 121736, unexpectedly inhibits TNF-mediated up-regulation of additional molecules on EC, including E-selectin (CD62E), VCAM-1 (CD106) and HLA-A,B,C. 736 siRNA transfection was not toxic for EC nor was there any evidence of an interferon response. 736 Transfection of EC blocked multiple early TNF-related signaling events, including activation of NF-kappaB. IL-1 activation of these same pathways was not inhibited. A unifying explanation is that 736 siRNA specifically reduced expression of mRNA encoding tumor necrosis factor receptor 1 (TNFR1) as well as TNFR1 surface expression. A sequence with high identity to the 736 antisense strand (17 of 19 bases) is present within the 3'UTR of human TNFR1 mRNA. An EGFP construct incorporating the 3'UTR of TNFR1 was silenced by 736 siRNA and this effect was lost by mutagenesis of this complementary sequence. Chemical modification and mismatches within the sense strand of 736 also inhibited silencing activity. In summary, an siRNA molecule selected to target ICAM-1 through its antisense strand exhibited broad anti-TNF activities. We show that this off-target effect is mediated by siRNA knockdown of TNFR1 via its sense strand. This may be the first example in which the off-target effect of an siRNA is actually responsible for the anticipated effect by acting to reduce expression of a protein (TNFR1) that normally regulates expression of the intended target (ICAM-1).

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Complementarity between TNFR1 and ICAM-1 does not activate a natural antisense transcript (NAT) activity. (A) HUVEC cell lines expressing EGFP/TNFR1 3′UTR chimeric constructs were treated for 24 h with increasing concentrations of TNF to induce ICAM-1 expression. ICAM-1 expression levels were determined by FACS using an ICAM-1-specific antibody. (B) HDMEC cell lines overexpressing E-selectin and ICAM-1 were tested to determine basal TNFR1 cell surface levels. Cells were immunostained with fluorescently labeled antibody specific for ICAM-1, TNFR1 and TNFR2 then analyzed by FACS. Representative of one of two independent experiments with similar results.
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Figure 10: Complementarity between TNFR1 and ICAM-1 does not activate a natural antisense transcript (NAT) activity. (A) HUVEC cell lines expressing EGFP/TNFR1 3′UTR chimeric constructs were treated for 24 h with increasing concentrations of TNF to induce ICAM-1 expression. ICAM-1 expression levels were determined by FACS using an ICAM-1-specific antibody. (B) HDMEC cell lines overexpressing E-selectin and ICAM-1 were tested to determine basal TNFR1 cell surface levels. Cells were immunostained with fluorescently labeled antibody specific for ICAM-1, TNFR1 and TNFR2 then analyzed by FACS. Representative of one of two independent experiments with similar results.

Mentions: Natural antisense transcripts (NAT), like miRNA and siRNA, are naturally occurring RNA species known to negatively regulate gene function by inhibition of transcription, RNA splicing and protein translation. In this report, we have identified and characterized an siRNA target sequence in the 3′UTR of TNFR1 which has 18 of 20 nt complementarity to a site within the coding sequence of human ICAM-1 (an additional homologous base pair between ICAM-1 and TNFR1 mRNA). Because of this complementary sequence relationship, we investigated whether TNFR1 transcript sequences might regulate ICAM-1 mRNA levels by an NAT mechanism, or vice versa. Because TNFR1 overexpression is toxic for EC we used our EGFP chimera transduced cell lines, which express steady state levels of EGFP/TNFR1 chimeric mRNA. These cells were treated with graduated amounts of TNF to induce ICAM-1 expression and cell surface ICAM-1 levels were measured by FACS to determine whether ICAM-1 levels were affected by TNFR1 sequences. All three cell lines responded to TNF treatment by up-regulating cell surface ICAM-1 levels. EGFP/TNFR1-3′UTR expressed slightly higher ICAM-1 levels than the other two lines (Figure 10A), and EFGP/736AS cells had nearly identical ICAM-1 protein levels as EGFP/Scramble. We conclude from this, and a similarly conducted experiment, that ICAM-1 protein expression is not negatively affected by the presence of complementary EGFP/TNFR1 chimeric mRNA sequences.Figure 10.


Knockdown of TNFR1 by the sense strand of an ICAM-1 siRNA: dissection of an off-target effect.

Clark PR, Pober JS, Kluger MS - Nucleic Acids Res. (2007)

Complementarity between TNFR1 and ICAM-1 does not activate a natural antisense transcript (NAT) activity. (A) HUVEC cell lines expressing EGFP/TNFR1 3′UTR chimeric constructs were treated for 24 h with increasing concentrations of TNF to induce ICAM-1 expression. ICAM-1 expression levels were determined by FACS using an ICAM-1-specific antibody. (B) HDMEC cell lines overexpressing E-selectin and ICAM-1 were tested to determine basal TNFR1 cell surface levels. Cells were immunostained with fluorescently labeled antibody specific for ICAM-1, TNFR1 and TNFR2 then analyzed by FACS. Representative of one of two independent experiments with similar results.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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Figure 10: Complementarity between TNFR1 and ICAM-1 does not activate a natural antisense transcript (NAT) activity. (A) HUVEC cell lines expressing EGFP/TNFR1 3′UTR chimeric constructs were treated for 24 h with increasing concentrations of TNF to induce ICAM-1 expression. ICAM-1 expression levels were determined by FACS using an ICAM-1-specific antibody. (B) HDMEC cell lines overexpressing E-selectin and ICAM-1 were tested to determine basal TNFR1 cell surface levels. Cells were immunostained with fluorescently labeled antibody specific for ICAM-1, TNFR1 and TNFR2 then analyzed by FACS. Representative of one of two independent experiments with similar results.
Mentions: Natural antisense transcripts (NAT), like miRNA and siRNA, are naturally occurring RNA species known to negatively regulate gene function by inhibition of transcription, RNA splicing and protein translation. In this report, we have identified and characterized an siRNA target sequence in the 3′UTR of TNFR1 which has 18 of 20 nt complementarity to a site within the coding sequence of human ICAM-1 (an additional homologous base pair between ICAM-1 and TNFR1 mRNA). Because of this complementary sequence relationship, we investigated whether TNFR1 transcript sequences might regulate ICAM-1 mRNA levels by an NAT mechanism, or vice versa. Because TNFR1 overexpression is toxic for EC we used our EGFP chimera transduced cell lines, which express steady state levels of EGFP/TNFR1 chimeric mRNA. These cells were treated with graduated amounts of TNF to induce ICAM-1 expression and cell surface ICAM-1 levels were measured by FACS to determine whether ICAM-1 levels were affected by TNFR1 sequences. All three cell lines responded to TNF treatment by up-regulating cell surface ICAM-1 levels. EGFP/TNFR1-3′UTR expressed slightly higher ICAM-1 levels than the other two lines (Figure 10A), and EFGP/736AS cells had nearly identical ICAM-1 protein levels as EGFP/Scramble. We conclude from this, and a similarly conducted experiment, that ICAM-1 protein expression is not negatively affected by the presence of complementary EGFP/TNFR1 chimeric mRNA sequences.Figure 10.

Bottom Line: An EGFP construct incorporating the 3'UTR of TNFR1 was silenced by 736 siRNA and this effect was lost by mutagenesis of this complementary sequence.We show that this off-target effect is mediated by siRNA knockdown of TNFR1 via its sense strand.This may be the first example in which the off-target effect of an siRNA is actually responsible for the anticipated effect by acting to reduce expression of a protein (TNFR1) that normally regulates expression of the intended target (ICAM-1).

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Yale University School of Medicine, New Haven, CT 06510, USA. paul.clark@yale.edu

ABSTRACT
Tumor necrosis factor (TNF) initiates local inflammation by triggering endothelial cells (EC) to express adhesion molecules for leukocytes such as intercellular adhesion molecule-1 (ICAM-1 or CD54). A prior study identified siRNA molecules that reduce ICAM-1 expression in cultured human umbilical vein EC (HUVEC). One of these, ISIS 121736, unexpectedly inhibits TNF-mediated up-regulation of additional molecules on EC, including E-selectin (CD62E), VCAM-1 (CD106) and HLA-A,B,C. 736 siRNA transfection was not toxic for EC nor was there any evidence of an interferon response. 736 Transfection of EC blocked multiple early TNF-related signaling events, including activation of NF-kappaB. IL-1 activation of these same pathways was not inhibited. A unifying explanation is that 736 siRNA specifically reduced expression of mRNA encoding tumor necrosis factor receptor 1 (TNFR1) as well as TNFR1 surface expression. A sequence with high identity to the 736 antisense strand (17 of 19 bases) is present within the 3'UTR of human TNFR1 mRNA. An EGFP construct incorporating the 3'UTR of TNFR1 was silenced by 736 siRNA and this effect was lost by mutagenesis of this complementary sequence. Chemical modification and mismatches within the sense strand of 736 also inhibited silencing activity. In summary, an siRNA molecule selected to target ICAM-1 through its antisense strand exhibited broad anti-TNF activities. We show that this off-target effect is mediated by siRNA knockdown of TNFR1 via its sense strand. This may be the first example in which the off-target effect of an siRNA is actually responsible for the anticipated effect by acting to reduce expression of a protein (TNFR1) that normally regulates expression of the intended target (ICAM-1).

Show MeSH
Related in: MedlinePlus