Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells

Abstract

RNA interference (RNAi) mediated by short interfering RNAs (siRNAs) is a widely used method to analyze gene function. To useRNAi knockdown accurately to infer gene function, it is essential to determine the specificity of siRNA-mediated RNAi. We have assessed the specificity of 10 different siRNAs corresponding to the MEN1 gene by examining the expression of two additional genes, TP53 (p53) and CDKN1A (p21), which are considered functionally unrelated to menin but are sensitive markers of cell state. MEN1 RNA and corresponding protein levels were all reduced after siRNA transfection of HeLa cells, although the degree of inhibition mediated by individual siRNAs varied. Unexpectedly, we observed dramatic and significant changes in protein levels of p53 and p21 that were unrelated to silencing of the target gene. The modulations in p53 and p21 levels were not abolished on titration of the siRNAs, and similar results were obtained in three other cell lines; in none of the cell lines tested did we see an effect on the protein levels of actin. These data suggest that siRNAs can induce nonspecific effects on protein levels that are siRNA sequence dependent but that these effects may be difficult to detect until genes central to a pivotal cellular response, such as p53 and p21, are studied. We find no evidence that activation of the double-stranded RNAtriggered IFN-associated antiviral pathways accounts for these effects, but we speculate that partial complementary sequence matches to off-target genes may result in a micro-RNA-like inhibition of translation.

Reconstituted influenza virus envelopes as an efficient carrier system for cellular delivery of small-interfering RNAs

“…Some nonspecific effects of siRNA at high concentrations were also observed for L2000-delivered control siRNA and have been described by others. 37,38 Results obtained with BHK21 cells were very similar to those shown for MDCK cells in Figure 8 (not shown).…”

Protein serine/threonine phosphatase-1 dephosphorylates p53 at Ser-15 and Ser-37 to modulate its transcriptional and apoptotic activities

“…First, a wild type of siRNA oligo and also a corresponding mutant (control) oligo were synthesized (Figure 3a). These oligos have been previously used by other researchers to silence p53 expression in other human cells (Scacheri et al, 2004). When the two oligos were separately introduced into HLECs for 72 h, silence of p53 expression by the wildtype siRNA oligo reached a level of 88% in comparison with the mutated oligo and a similar level when compared with the nontransfected HLECs (Figure 3b).…”

Use of interfering RNA to investigate the role of endogenous gastrin in the survival of gastrointestinal cancer cells

“…The effects of the gastrin siRNA appear to be specific. The possibility of off-target effects (Scacheri et al, 2004) and induction of the interferon response (Sledz et al, 2003) has been suggested in the literature. A BLAST search was included in the design of the siRNAs used in this study and no matches to other genes were identified.…”