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