miR-25-3p Modulates Tumor Aggressiveness and Ferroptosis Escape in T24 Bladder Cancer Cells In Vitro

<b>Background/Objectives</b>: Urothelial bladder carcinoma (UBC) is one of the most prevalent malignancies worldwide, and efforts have intensified to identify molecular markers that improve the prognosis and reduce treatment costs. Among the regulators of tumor behavior, microRNAs (miRNAs) have emerged as promising biomarkers for cancer diagnoses and treatment. The modulation of miR-25-3p has been associated with pancreatic, colorectal, and lung cancers; its role in UBC remains poorly explored. In this study, we investigated the effects of miR-25-3p modulation in a high-grade and muscle-invasive bladder cancer (MIBC) cell line (T24), using in vitro functional assays and bioinformatics approaches. <b>Results:</b> Bioinformatics analyses using TCGA-BLCA datasets revealed that miR-25-3p is upregulated in tumor tissues compared to non-tumor tissues, prompting an investigation into its molecular targets and related pathways. The transfection of T24 cells with an miR-25-3p mimic and inhibitor led to respective overexpression (11.16-fold) and downregulation (-2.82-fold) compared to the negative control. Functionally, miR-25-3p overexpression increased cell proliferation, viability, and migration, while its inhibition decreased the cell migration capacity. A gene expression analysis revealed that miR-25-3p overexpression resulted in the downregulation of <i>TP53</i>, <i>AIFM1</i>, <i>NFE2L2</i>, <i>TFRC</i>, <i>ACSL4</i>, <i>SLC7A11</i>, and <i>SLC3A2</i>, whereas <i>MMP9</i>, <i>MMP11</i>, and <i>GPX4</i> were upregulated, suggesting a role in both migration and ferroptosis regulation. In the inhibitor group, increased <i>SLC3A2</i> and decreased <i>MMP11</i> expression further supported this connection. Our results using an in vitro model for MIBC with the transfection of T24 cells suggest that miR-25-3p influences key pathways involved in oxidative stress and cell death, promoting a more aggressive tumor phenotype. <b>Conclusions:</b> The modulation of miR-25-3p impacts the behavior of T24 bladder cancer cells and may indicate its role in disease progression. Our results underscore the potential of miR-25-3p as a prognostic biomarker and support further studies considering its therapeutic relevance in managing high-grade and muscle-invasive bladder cancer.