Comparing Linear and Circular RNA Enrichment in HeLa Cell-derived EVs

• Percival Tran, Applied Molecular Biology & Biotechnology, University of Delaware

• Mona Batish, Department of Medical and Molecular Sciences, University of Delaware

CircRNAs are strands of RNA naturally found in eukaryotes that are covalently closed into continuous loops by the process of back-splicing. While some circRNAs have been found to play important roles in regulating protein function and inhibition, others have been speculated to affect the progression of certain diseases and cancers. CircRNAs can be found both inside and outside of eukaryotic cells. Extracellular vesicles (EVs) are naturally occurring vesicles secreted by cells, composed of lipids, nucleic acids, and proteins encapsulated by a lipid bilayer. EVs are useful diagnostic tools because they are abundantly found in bodily fluids and contain many disease biomarkers including circRNAs. In this experiment, HeLa cells expressing linear and circular forms of GFP RNA were used to explore the hypothesis that circRNAs are more enriched in EVs than their linear counterparts. HeLa cells were infected with viruses containing either pCS-CG, which encodes linear GFP, or a circular GFP (cGFP) construct, which encodes a circular form of GFP; wild-type HeLa cells were used as a control. RNA was isolated from the three different HeLa cell variants as well as from EVs collected from those cells. RT-PCR was then performed to compare the enrichment of both linear and circular GFP constructs in EVs compared to their cellular levels. Band intensity was quantified to compare the enrichment in different samples. Results showed that circGFP was more enriched in EVs compared to linear GFP. These results are consistent with previous reports that found higher enrichment of circRNAs in EVs compared to their linear counterparts.