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

Proc Natl Acad Sci U S A. 2004 Feb 17;101(7):1892-7. doi: 10.1073/pnas.0308698100. Epub 2004 Feb 9.


RNA interference (RNAi) mediated by short interfering RNAs (siRNAs) is a widely used method to analyze gene function. To use RNAi 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 RNA-triggered 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.

MeSH terms

  • 2',5'-Oligoadenylate Synthetase / genetics
  • Animals
  • Blotting, Western
  • Cell Line
  • Cyclin-Dependent Kinase Inhibitor p21
  • Cyclins / genetics
  • Cyclins / metabolism
  • Gene Expression Regulation*
  • HeLa Cells
  • Humans
  • Interferons / physiology
  • Proto-Oncogene Proteins / genetics*
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • RNA, Small Interfering / genetics
  • RNA, Small Interfering / metabolism*
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sequence Homology
  • Substrate Specificity
  • Transfection
  • Tumor Suppressor Protein p53 / genetics
  • Tumor Suppressor Protein p53 / metabolism


  • CDKN1A protein, human
  • Cyclin-Dependent Kinase Inhibitor p21
  • Cyclins
  • MEN1 protein, human
  • Proto-Oncogene Proteins
  • RNA, Messenger
  • RNA, Small Interfering
  • Tumor Suppressor Protein p53
  • Interferons
  • OAS1 protein, human
  • 2',5'-Oligoadenylate Synthetase