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 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.

Fig. 1.

Western blot and quantitative RT-PCR analyses of siRNA transfected cells. (a) Western blot analysis of HeLa cells transfected with 10 different MEN1-siRNAs show significant differences in the levels of p21 and p53 that are independent of menin levels. Titration of the siRNA did not abolish these effects. p53-siRNA was used as a positive control for silencing. LacZ- and CAT-siRNAs are negative controls for menin silencing but also show changes in the levels of menin (LacZ) and p21 (LacZ and CAT). Actin serves as a loading control. (b) Western blot analyses of CaSki, SiHa, and MCF7 cells also show dramatic differences in the levels of p53 and p21 that are independent of menin levels. (c) Quantitative PCR assays showing the percent of MEN1, p53, and p21 mRNA expression relative to mock transfected cells. Note that the relative differences in mRNA expression are small (<2-fold) relative to the changes seen on Western blot.

Fig. 2.

Quantitative RT-PCR analysis of OAS1. OAS1 induction by all 13 siRNAs is within 3-fold, which is far less than that reported on activation of an IFN response.