An RNA aptamer provides a novel approach for the induction of apoptosis by targeting the HPV16 E7 oncoprotein

PLoS One. 2013 May 30;8(5):e64781. doi: 10.1371/journal.pone.0064781. Print 2013.

Abstract

Background: Human 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 findings: This 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/significance: This 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apoptosis*
  • Aptamers, Nucleotide / genetics
  • Aptamers, Nucleotide / metabolism*
  • Base Sequence
  • Cell Line, Tumor
  • Cell Proliferation
  • Cell Transformation, Viral
  • Humans
  • Papillomavirus E7 Proteins / metabolism*
  • Retinoblastoma Protein / metabolism
  • Transfection

Substances

  • Aptamers, Nucleotide
  • Papillomavirus E7 Proteins
  • Retinoblastoma Protein
  • oncogene protein E7, Human papillomavirus type 16

Grant support

This work was partly supported by Yorkshire Cancer Research (L333) (http://www.yorkshirecancerresearch.org.uk/) and by a BBSRC studentship to CN. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.