Identification of circCIAO1(5) and circMALAT1 as Novel Potential Biomarkers for Bladder Cancer Monitoring Based on the Binding to miR-101-3p

Background and Objectives: Bladder cancer (BCa) is characterized by high rates of recurrence and progression, underscoring the need for reliable non-invasive biomarkers. Circular RNAs (circRNAs) are covalently closed non-coding RNAs generated by back-splicing and are stable in biological fluids, including urine. Increasing evidence implicates circRNAs in BCa pathogenesis. However, identification of clinically relevant circRNAs remains challenging. This study aimed to streamline circRNA selection and identification of functional urinary circRNAs for potential use as biomarkers for BCa monitoring. Methods: Using a database-screening approach, we identified circRNAs with high predicted affinity (TDMD score > 1.1) to miR-101-3p (a tumor-suppressive microRNA in BCa). In addition, candidate circRNAs were prioritized based on the following: (i) derivation from genes involved in BCa tumorigenesis; and (ii) origination from exonic or long non-coding RNA sequences. The potential contribution of Argonaute-2 (Ago2) binding sites to circRNA activity or potential usage as biomarker was also evaluated. Expression levels were assessed in urine samples and BCa cell lines, and functional relevance was examined using molecular and cellular assays. Results: circCIAO1(5) and circMALAT1 fulfilled prioritization criteria and exhibited distinct Ago2-binding site profiles. Both circRNAs were upregulated in urine from BCa patients and in aggressive BCa cell lines and showed differential expression between remission and recurrent disease. CircCIAO1(5) demonstrated higher-affinity binding to miR-101-3p, while both circRNAs interacted with miR-101-3p and Ago2. Functional assays revealed enhanced proliferation, motility, and invasion upon circRNA overexpression, consistent with miR-101-3p sequestration and reduction in depression of its target oncogene—EZH2. Conclusions: circCIAO1(5) and circMALAT1 are promising candidates as urinary biomarkers for noninvasive BCa monitoring, illustrating the value of bioinformatics-guided determination of circRNA as potential biomarkers and significance of circRNA-mediated regulatory mechanisms in BCa biology.