Noninvasive identification of lineage-specific circular RNA for ER-positive breast cancer.

Abstract

3543Background: Non-invasive testing in plasma using RNA biomarkers has been limited by exoribonuclease-mediated degradation of RNA. Circular RNA (circRNA) are covalently closed RNA structures that resist this degradation due to their circular structure. Therefore circRNA are more stable than their linear counterparts. CircRNA are formed by alternative backsplicing of the 3’ end of a downstream exon to the 5’ end of an upstream exon. Here, we propose a novel method for non-invasive identification of circRNA and demonstrate circularized forms of several lineage and cancer specific targets for estrogen receptor-positive breast cancer. Methods: Capture RNA sequencing on cancer tissue was previously performed to determine the relative expression of potential circRNA isoforms in breast cancer patients. These isoforms as well as those predicted by intron length were screened using a quantitative PCR-based assay on ER-positive breast cancer cells. RNA extracted from breast cancer cells are exposed to ribonuclease R to demonstrate stability of circRNA. CircRNA derived from targets with known universal expression are used as positive controls as well as for analysis on plasma. Results: We identify the circRNA isoforms with highest expression for five genes, including ESR1, that are differentially expressed in ER-positive breast cancer compared to other cancers and normal breast tissue. We determine that the circRNA corresponding to all five targets is specifically expressed in breast cancer cell lines with at least 1000-fold higher expression than in non-ER positive breast cancer cell lines. We demonstrate that the highest expressing circRNA isoforms are resistant to degradation by ribonuclease R, whereas corresponding linear mRNA is susceptible. We also demonstrate the presence and stability of positive control circRNA in plasma from patients without cancer. Conclusions: CircRNA are promising biomarkers for early non-invasive detection of cancer due to their stability in plasma. This assay reliably detects ER-positive breast cancer specific circRNA, and exoribonuclease resistance has been validated. Application of this diagnostic assay to plasma from breast cancer patients is underway.