Design, Construction, and Screening of an shRNA Library Targeting Human Circular RNAs
Islam, MD Fahmid 1989-
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Recent advances in next-generation sequencing (NGS) methods and computational analyses have identified a large class of non-polyadenylated RNA molecules. Further, analysis of these RNAs revealed several unexpected junctions, which do not map to mRNAs, leading to the discovery of circular RNAs (circRNAs). Unlike linear RNAs, circRNAs have no ends and are not sensitive to exoribonucleases, endowing circRNAs with a longer half-life. This enhanced stability has prompted the study of circRNAs as cancer biomarkers. While expression studies are becoming widely used to profile circRNAs in multiple cancers, there are no genome-wide tools available to decrease their levels in cells. Methods that investigate the role of circRNAs through measuring their expression are prone to artifacts as bypassing transcription termination results in RNA concatamers. To better characterize the function of circRNAs, we developed a novel pooled library of ~ 15,000 shRNAs targeting ~ 5,000 circRNAs. We performed a loss-of-function screen with the circRNA shRNA library in a colorectal cancer cell line to systematically identify circRNAs that were required for cell proliferation and survival. During the construction and validation of this library, we also developed a method to improve NGS quality by reducing sequencing failure due to shRNA hairpin and/or heteroduplex formation. Using this approach, we identified and validated several circRNAs essential for the survival of colorectal cancer cell lines. We believe that these essential circRNAs will provide an opportunity to understand cancer biology in a more detailed way, and to design effective cancer therapeutics and diagnostics.
DegreeMaster of Science (M.Sc.)
CommitteeWilson, Joyce; Lee, Jeremy; Geyer, Ron; Vizeacoumar, Franco; Kusalik, Anthony; Leary, Scot
Copyright DateJuly 2018
circular RNA, cancer, essentiality , screen, next-generation sequencing