Development of androgen receptor messenger RNA targeted molecular beacons for use in the study of prostate cancer progression
Glick, Cindy Jennifer
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Messenger RNA (mRNA) posttranscriptional regulation has been implicated in the development and/or progression of several diseases including many types of cancer, rheumatoid arthritis, vascular disease, and Alzheimer's disease. Differential regulation of Androgen Receptor (AR) mRNA has been associated specifically with prostate cancer progression. In this thesis, molecular beacons were developed to allow for the detection of the expression and localization of AR mRNA in live prostate cancer cells. These beacons were then applied as a tool for studying how AR mRNA regulation is involved in prostate cancer growth and advancement. Two AR mRNA targeted beacons were designed and tested in solution and in live cells to determine their functionality. The beacon-based approach for AR mRNA detection was then optimized through the use of the two beacons in tandem and alteration of their backbone chemistry. A series of validation tests were performed on these beacons, including testing their abilities to: 1) produce a feasible localization pattern, 2) discriminate between AR positive (AR+) and AR negative (AR-) prostate cancer cell lines and 3) follow stimulus-induced changes in AR mRNA expression. Based on these results, a dual chimeric beacon approach was selected to determine the role of AR mRNA regulation in two systems that represent important stages in prostate cancer growth and progression: 1) hormone stimulation of androgen-dependent prostate cancer cells and 2) progression of androgen-dependent prostate cancer cells to the androgen-independent state. Our results suggest that changes in AR mRNA expression, organization, and localization may be indicative of molecular mechanisms involved in these critical transitions associated with prostate cancer progression. Taken together, this work provides a feasibility study for visualizing changes in AR mRNA state as a diagnostic measure for evaluating the aggressiveness of the disease and demonstrates the possible utility of therapeutically targeting AR mRNA regulation in order to prevent prostate cancer advancement.