MicroRNA Signatures of Prostate Cancer Spheroids in Microfluidic Culture Under Hormone-Deprivation Conditions

Background: Prostate cancer (PCa) is prevalent in men over 65 and requires effective clinical management. Standard PCa therapies often offer positive outcomes; however, its castration-resistant form (CRPC) is aggressive and associated with poor prognosis. The objective of this study is to characterize the microRNA profiles associated with the PCa to CRPC transition using a microfluidic PCa model. Methods: LNCaP-derived hormone-sensitive PCa spheroids were cultured for 30 days under recirculating flow conditions mimicking hormone deprivation. Total RNA was isolated from the spheroids and perfusate at Day 5 and Day 30. Exosomal microRNAs were profiled by miRNA-seq. Differentially expressed miRNAs were used for target prediction across multiple databases, and gene set enrichment analysis (GSEA) was performed to identify pathways affected during prolonged hormone deprivation. Results: Sustained hormone deprivation induced a shift in microRNA expression. Tumor-suppressive miRNAs were broadly reduced. To evaluate functional consequences, predicted targets were compiled for all regulated miRNAs. For the 33 intracellular miRNAs downregulated on Day 30, 430 genes were predicted as targets for at least 16 of these miRNAs, revealing strong convergence on shared regulatory pathways. Thirty-five genes overlapped with predicted targets of the single upregulated miRNA and were removed, yielding a refined set of 395 unique genes used for GSEA. Overall, the neuronal differentiation pathways observed reflect early features of a neuroendocrine-like phenotype. Conclusions: This microfluidic PCa model captures early molecular events associated with progression toward CRPC. It provides a controlled system for studying disease evolution and supports the development of more precise therapeutic and diagnostic strategies.