Identification of circCIAO1(5) and circMALAT1 as Novel 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 re-currence 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 im-plies circRNAs in BCa pathogenesis; however, identification of clinically relevant circRNAs remains labor-intensive. This study aimed to streamline circRNA selection and identify functionally relevant urinary circRNAs in BCa. Methods: Using a database-screening ap-proach, we identified circRNAs with high predicted affinity to miR-101-3p, a tu-mor-suppressive microRNA in BCa. Candidate circRNAs were prioritized based on: (i) strong miR-101-3p binding potential; (ii) derivation from genes involved in BCa tumor-igenesis; and (iii) origination from exonic or long non-coding RNA sequences. The po-tential contribution of Argonaute-2 (Ago2) binding sites to circRNA–miRNA complex sta-bility 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 all 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 RNA immunoprecipitation confirmed interactions of both circRNAs with miR-101-3p and Ago2. Functional assays revealed enhanced proliferation, motility, and invasion upon circRNA expression, consistent with miR-101-3p sequestration and derepression of miR-101-3p target oncogene-EZH2. Conclusions: circCIAO1(5) and circMALAT1 represent promising urinary biomarkers for BCa, illustrating the value of bioinformatics-guided circRNA discovery and significance of circRNA-mediated regulatory mechanisms in BCa biology.