Background <p>The development of resistance to anti-androgen therapies, such as bicalutamide (Bic), poses a significant challenge in the treatment of prostate cancer (PCa), and the intercellular complex mechanisms are difficult to comprehend. Emerging evidence suggests that stem cells and their secreted exosomes (Exo) contribute to tumor microenvironment-mediated drug resistance. However, the role of these vesicles in promoting bicalutamide resistance in prostate cancer remains elusive. This work aimed to clarify the mechanism by which exosomes from bicalutamide-stimulated mesenchymal stem cells (Bic-MSCs-Exo) provide resistance to bicalutamide in prostate cancer.</p> Methods <p>This work aimed to clarify the function of exosomes produced from Bic-MSCs in bicalutamide resistance in PCa and its molecular underpinnings. The impact of Bic-MSCs supernatants and their exosomes on cell proliferation, clone formation, migratory capacity, and apoptosis was assessed by the co-culturing of these supernatants and exosomes with PCa cells to elucidate their pro-resistance phenotype. Additional short RNA sequencing of exosomes, together with bioinformatics and single-cell data analysis, was conducted to identify and characterize the pivotal molecule lncRNA SILC1. The downstream regulatory network was validated by dual-luciferase reporter assays, functional rescue and loss-of-function tests, quantitative PCR, and Western blotting; the molecular processes were confirmed at both the in vivo and clinical levels using an organoid model and clinical samples analyzed via FISH.</p> Results <p>We found that Bic-MSCs-derived exosomes (Bic-MSCs-Exo) promote prostate cancer (PCa) cell survival and drug resistance. Using sequencing and quantitative PCR (qPCR) analyses, we found that exosomes carry the drug-resistant molecule <i>SILC1</i>, which binds <i>miR-577</i> and functions as a competing endogenous RNA (ceRNA). Combining multi-database analyses with experimental data, we confirmed that <i>miR-577</i> targets and inhibits <i>RHOA</i> expression. Notably, high expression levels of <i>RHOA</i> are associated with increased proliferation, invasion, and enhanced clonogenic ability in prostate cancer cells. Furthermore, we demonstrated that the knockdown of <i>SILC1</i> leads to the downregulation of <i>RHOA</i>. In organoid models of hormone-sensitive prostate cancer (HSPC) and Castration-Resistant Prostate Cancer (CRPC), it was found that RHOA was considerably overexpressed in CRPC. Additionally, a fluorescence in situ hybridization (FISH) study of clinical specimens demonstrated a link among the expression of SILC1, miR-577, and RHOA.</p> Conclusions <p>The discovery of the lncRNA <i>SILC1</i> /<i>RHOA</i> as a promising marker of PCa resistance sheds light on the molecular pathogenesis of PCa resistance and provides potential therapeutic targets for the disease.</p>