Aptamer-Based Discovery of Neuropilin-2 as a Potential Biomarker and Therapeutic Target in Bladder Cancer

Bladder cancer (BLCA) remains a clinically challenging malignancy with high recurrence rates and limited molecular targeting options, highlighting the critical need to identify novel biomarkers and develop targeted molecular tools for improved diagnosis and therapy. Aptamers, owing to their high affinity and specificity, represent promising molecular tools for bladder cancer detection and targeted intervention. Using Cell-SELEX against BLCA SCaBER cells combined with counterselection against normal SV-HUC-1 cells, we successfully obtained a DNA aptamer, ss2, which was subsequently optimized to yield a truncated variant, ss2-2. The resulting aptamer ss2-2 exhibited a strong binding affinity and high specificity for BLCA cells. Through aptamer pull-down coupled to mass spectrometry, the molecular target of ss2-2 was identified as neuropilin-2 (NRP2), a transmembrane glycoprotein. This interaction was further validated through siRNA-mediated knockdown and binding correlation analyses across multiple cell lines. Molecular docking revealed a stable interaction interface between ss2-2 and NRP2. Clinically, immunohistochemistry analysis of a BLCA tissue microarray demonstrated significantly elevated NRP2 expression in BLCA tissues, which correlated with an advanced tumor stage. Functionally, NRP2 knockdown suppressed BLCA cell proliferation, colony formation, and migration and regulated the AMPK, MAPK/ERK, and STAT3 signaling pathways. Collectively, these results identify ss2-2 as a high-affinity aptamer targeting NRP2 and underscore the potential of NRP2 as a promising diagnostic biomarker and therapeutic target in bladder cancer, providing a robust foundation for the development of aptamer-based molecular diagnostic and targeted therapeutic strategies.