Correlation between circular-RNAs contained in extracellular vesicles with BRCA status in ovarian cancer cells

Introduction: Ovarian cancer (OC) is a common malignancy with highmortality. Highlighting the mechanisms of OC progression and discovery of new biomarkersmay improve OC diagnosis and therapy.OC is often caused by germ-line mutations in BRCA1 genes. Circular RNAs (circRNAs) are a class of novel non-coding RNAs that undergoe back-splicing and form a covalently closed loop. circRNAs have regulative roles in cancer. Extracellular vesicles (EVs) are natural intercellular shuttles for circRNAs. Our study aims to correlate circRNAs shuttled by OC-EVs with BRCA mutational status. Methods: Human OC cell lines UWB1.289 (BRCA1-null), UWB1.289 + BRCA1 (BRCA1-restored) were cultured in RPMI/HUMEC medium (3% FBS); ES-2 (BRCA wild type) was cultured in McCoy’s 5A medium (10% FBS). EVs were isolated by ultracentrifugation from both UWB cell lines and characterized according to MISEV 2023. To analyse CircRNAs in UWB cells and EVs, RNA was extracted with a commercial kit, and complementary DNA was synthesized by reverse transcription. circRNA was evaluated using divergent primers by RT-qPCR with SYBR Green. GAPDH was used as a reference. Relative changes in circRNA expression were determined by the ΔΔCt method. The effects of UWB-EVs on ES-2 cell proliferation and migration were evaluated byMTS/CCK8 and wound healing assay, respectively. Results: EVs were isolated from both UWB cell lines and characterized: they appear intact and rounded (TEM), with a mean size ranging around 160–190 nm (NTA), and are positive for CD63, CD9, TSG-101 and negative for CNX (WB). A selection of circRNAs (CDR1AS, HIPK2, HIPK3, FAM120a, ITCH, AFF1, MUC16) was tested on cells and EVs in both UWB cell lines. Among these circRNAs, MUC16 and FAM120a were represented differently in UWB cells and EVs. Notably, UWB-EVs were able to modulate ES-2 proliferation and migration. Summary/Conclusion: Multiple genes with germLine mutations have been implicated in hereditary OC. Our results indicate that, in OC cell lines, circRNAs contained in EVs could be linked to BRCA mutational status andOC progression. This study confirms the potential of circRNAs shuttled by EVs as biomarkers for OC diagnosis and therapeutic targets. Funding: Project funded from Program PRIN 2022 under grant agreement N◦20224XB79P by the European Union, nextgeneration EU, PNRR, M.4-C2-1.1.