Selection of Aptamers for CED-9/Bcl-2 Family Cell Death Regulations and Their Application in Study of Apoptosis Regulation and Drug Design for Breast Cancer

摘要

Apoptosis has been found to be conserved from C. elegans to humans, suggesting that novel means developed in C. elegans to modulate apoptosis may also be applied to humans for therapeutic treatments of diseases caused by abnormal apoptosis (e.g. cancer, autoimmune diseases, neurodegenerative diseases, etc). In this study, we employed the technique of SELEX to identify small RNA aptamers with high binding specificity and affinity for key cell death regulators, including CED-9 and CED-4 from C. elegans and Bcl-2 and Bcl- xL from humans. So far, we have isolated five RNA aptamers that bind CED-9 with Kds ranging from 5 nM to 10 nM. These aptamers can be categorized into three groups based on their secondary structures and appear to bind to overlapping sites on CED-9. Furthermore, these CED-9 aptamers can form a ternary complex with CED-9 and EGL-1, but not with CED-9 and CED-4, suggesting that these aptamers and EGL-l bind to different surface regions on CED-9. When over expressed in C. elegans touch receptor neurons, these aptamers can induce ectopic neuronal apoptosis. Importantly, the ectopical cell killing induced by the aptamers is inhibited by a loss-of-function mutation in a key cell-killing gene (ced-3) encoding the caspase, suggesting that these aptamers kill cells through the normal apoptotic pathway. We have conducted several rounds of SELEX experiments on CED-4 and Bcl-xL and obtained candidate molecules with increasing binding affinity in vitro to these two proteins. Our studies suggest that RNA aptamers can be used to modulate apoptosis in vivo and can potentially be used to develop drugs to treat cancers caused by abnormal apoptosis, including breast cancers.