Introduction: Prostate cancer (PCa) innervation contributes to the progression of PCa. However, the precise impact of innervation on PCa cells is still poorly understood. We have recently shown that muscarinic receptors promote castration-resistant growth of PCa through FAK-YAP signaling axis. However, mechanisms of upregulation of muscarinic receptors leading to castration-resistant growth of PCa cells were unclear. Methods: Small RNA-sequencing and bioinformatics screening were used to identify candidate microRNAs to regulate muscarinic receptors. Luciferase reporter assay was performed to see direct regulation of muscarinic receptors by microRNAs. Western blot and qRT-PCR verified the effect of the microRNA on downstream YAP signaling. Cell proliferation and sphere assay demonstrated the effect of microRNA on the muscarinic-receptor-driven castration-resistant growth of PCa. Results: Small RNA-seq showed that 255 microRNAs were significantly downregulated under castrated condition in LNCaP cells. Using bioinformatic approach, microRNA-15b-5p was found to be one of the candidate microRNAs that directly regulate m3. microRNA-15b-5p was confirmed to downregulate with castrate condition, in contrast to the upregulation of muscarinic receptor 3 (m3) with castration in vitro. Indeed, microRNA-15b-5p was downregulated in castration-resistant PCa (CRPC) compared with hormone-sensitive PCa (HSPC) specimens. Moreover, microRNA-15b-5p directly binds to m3, and microRNA-15b-5p inhibited YAP activation which was induced by m3 stimulation. Furthermore, microRNA-15b-5p abolished castration-resistant growth of PCa cells which was induced by m3 stimulation, in 2D cell proliferation assay and 3D cell sphere assay. Conclusions: microRNA-15b-5p/m3/YAP signaling axis is a novel pathway which promotes castration-resistant growth of PCa. Our findings provide novel mechanisms of muscarinic-signal-driven CRPC progression. SOURCE OF Funding: JSPS KAKENHI Grant Number: 21K16753
