miRNAs Delivered by Prostate Cancer Extracellular Vesicles Coordinate the Regulation of Bone Metastasis

Abstract Background We previously reported that extracellular vesicles derived from osteoblastic, osteoclastic, and mixed prostate cancer (PCa) cells promote osteoclast differentiation and inhibit osteoblast differentiation via the transfer of miR-92a-1-5p. However, at the same time, osteoblastic miRNAs also exist in PCa extracellular vesicles. In the present study, we focused on discovering (1) the roles of miR-375 and miR-148a-3p delivered by PCa extracellular vesicles in bone homeostasis and bone metastasis, and (2) an explanation for the co-existence of osteoblastic miRNAs and osteoclastic miRNAs in prostate cancer extracellular vesicles. Methods Conditional medium, miRNA mimics, and miRNAs overexpressed lentiviruses were employed to discover the roles of miR-375 and miR-148a-3p delivered by PCa extracellular vesicles in bone homeostasis and tumor growth. Target gene prediction and siRNAs were employed to discover the target gene(s) for miR-375 and miR-148a-3p. Droplet digital PCR (ddPCR) of the PCa extracellular vesicle miRNAs were utilized to evaluate miRNA expression at different metastatic phases. Results Conditional medium from prostate cancer cell culture promoted osteoblast differentiation, as confirmed by ALP staining and Alizarin red staining. miR-375 and miR-148a-3p promoted osteoblast differentiation in vitro by reducing KLF4 expression, associated with increased osteoblast function as shown by ALP staining, Alizarin red staining, and Alp mRNA expression. In vivo , miR-375 and miR-148a-3p overexpressing MDA PCa 2b cells promoted osteoblastogenesis and tumor growth, which were confirmed by micro computed tomography and in vivo imaging. siRNA targeting KLF4 similarly enhanced osteoblast function and tumor cell proliferation. Based on the miRNA ddPCR data for PCa extracellular vesicles, osteoclastic and osteoblastic miRNAs existed in different bone metastatic phases. Conclusions These findings suggest that miR-375 and miR-148a-3p delivered by PCa extracellular vesicles regulate osteoblast function and tumor growth via targeting KLF4 . Osteoclastic miR-92a-1-5p is active during the early phases of bone metastasis, while osteoblastic miR-375 and miR-148a-3p function during the late phases.