An RNA Aptamer Provides a Novel Approach for the Induction of Apoptosis by Targeting the HPV16 E7 Oncoprotein

Abstract

BackgroundHuman papillomavirus 16 (HPV16) is a high-risk DNA tumour virus, which is a major causative agent of cervical cancer. Cellular transformation is associated with deregulated expression of the E6 and E7 oncogenes. E7 has been shown to bind a number of cellular proteins, including the cell cycle control protein pRb. In this study, RNA aptamers (small, single-stranded oligonucleotides selected for high-affinity binding) to HPV16 E7 were employed as molecular tools to further investigate these protein-protein interactions.Methodology/Principal FindingsThis study is focused on one aptamer (termed A2). Transfection of this molecule into HPV16-transformed cells resulted in inhibition of cell proliferation (shown using real-time cell electronic sensing and MTT assays) due to the induction of apoptosis (as demonstrated by Annexin V/propidium iodide staining). GST-pull down and bead binding assays were used to demonstrate that the binding of A2 required N-terminal residues of E7 known to be involved in interaction with the cell cycle control protein, pRb. Using a similar approach, A2 was shown to disrupt the interaction between E7 and pRb in vitro. Furthermore, transfection of HPV16-transformed cells with A2 appeared to result in the loss of E7 and rise in pRb levels, as observed by immunoblotting.Conclusions/SignificanceThis paper includes the first characterisation of the effects of an E7 RNA aptamer in a cell line derived from a cervical carcinoma. Transfection of cells with A2 was correlated with the loss of E7 and the induction of apoptosis. Aptamers specific for a number of cellular and viral proteins have been documented previously; one aptamer (Macugen) is approved for clinical use and several others are in clinical trials. In addition to its role as a molecular tool, A2 could have further applications in the future.

Use of Aptamers as Diagnostics Tools and Antiviral Agents for Human Viruses

“…In an interesting study, Nicol et al selected an RNA aptamer, termed A2, with high affinity to HPV-16 E7 [125]. Transfection of this aptamer into HPV-16-transformed cells resulted in induction of apoptosis and consequent inhibition of cell proliferation.…”

“…Transfection of this aptamer into HPV-16-transformed cells resulted in induction of apoptosis and consequent inhibition of cell proliferation. In addition, the authors demonstrated that A2 bound to the N-terminal residues of E7 required for interaction with pRb and that, consequently, A2 disrupted the interaction between E7 and pRb in vitro [125]. Furthermore, the authors demonstrated that A2 enhanced E7 localization in the ER and that the A2-mediated reduction of E7 was not associated with proteasomal degradation suggesting that A2 perturbs normal E7 trafficking through promoting E7 ER retention [126].…”

Experimental and Computational Considerations in the Study of RNA-Binding Protein-RNA Interactions

“…The single-stranded nature of RNA allows an RNA molecule to form a wide variety of secondary, tertiary, and even quaternary structures that enable a protein to not only interact with the ribonucleotide sequence, but also the nucleotide backbone in unique forms governed by the structure of the RNA. Indeed, RNA aptamers can be evolved in vitro that strongly interact with small molecules such as fluorophores 6 or specific proteins of interest 7,8 .…”

Application of Nucleic Acid Aptamers to Viral Detection and Inhibition

“…The same research group also isolated RNA aptamers against E7 oncoprotein that were able to disturb the E7-pRb interaction by targeting E7 for degradation and showed that one of them (A2) was able to inhibit cellular proliferation by inducing apoptosis in SiHa cervical carcinoma cells [54]. This effect was specific to HPV-16 transformed cells because it was not observed in HPV-free or HPV-18 cell lines [55]. Specific apoptosis induction of RNA aptamers targeting E6 and E7 oncoproteins suggests that these aptamers could have further applications in the future as therapeutic moieties.…”