Milk Extracellular Vesicle–mediated Delivery of siPRNP Suppresses Growth and Wound Closure in PC-3M Prostate Cancer Cells

<h3>Background/Aim:</h3> Metastatic prostate cancer remains difficult to treat, in part because tumor cells maintain high growth and motility. This study examined whether inhibition of hsa-miR-4516 reduces PRNP/PrP^C expression and decreases cell number and wound closure in PC-3M cells, and whether PRNP knockdown delivered by milk extracellular vesicles (milk EVs) produces similar effects. <h3>Materials and Methods:</h3> Expression of hsa-miR-4516 was compared between LNCaP-LN3 and PC-3M cells by quantitative real-time PCR. PC-3M cells were transfected with a miR-4516 inhibitor; PRNP mRNA was quantified by quantitative real-time PCR; and PrP^C and cell-cycle proteins (CDK2, CDK4, cyclin D1, cyclin E) were assessed by western blotting together with cell number quantification. PRNP-targeting siRNAs were screened, and siRNA #3 was loaded into milk EVs and applied to PC-3M cells (milk EV–siPRNP). PrP^C and cell-cycle proteins were examined by western blotting. Single-cell outgrowth was evaluated by limiting dilution in 96-well plates followed by 6-day culture, and motility was assessed by wound-healing assay (24 h). <h3>Results:</h3> PC-3M cells showed higher hsa-miR-4516 expression and higher PrP^C protein levels than LNCaP-LN3 cells. In PC-3M cells, miR-4516 inhibition reduced PRNP mRNA and PrP^C protein levels, decreased CDK4, cyclin D1, and cyclin E expression, and reduced cell numbers. Milk EV–siPRNP decreased PrP^C and reduced CDK2, CDK4, cyclin D1, and cyclin E. In addition, milk EV–siPRNP suppressed single-cell outgrowth during 6-day culture and delayed wound closure in the wound-healing assay. <h3>Conclusion:</h3> In PC-3M cells, inhibition of hsa-miR-4516 or PRNP knockdown reduced PRNP/PrP^C and cell-cycle regulators, decreased cell accumulation, and delayed wound closure. Milk EV–siPRNP achieved PRNP knockdown and reduced single-cell outgrowth and wound closure in PC-3M cells.