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Milk Extracellular Vesicle-mediated Delivery of siPRNP Suppresses Growth and Wound Closure in PC-3M Prostate Cancer
Jeongkun Lee1, Junyoung Yoon1, Jae Young Lee1
1Department of Biomedical Sciences, College of Medicine, and Program in Biomedical Science & Engineering, Inha University, Incheon, Republic of Korea.
Background/Aim:
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/PrPC 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.
Materials And Methods:
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 PrPC 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). PrPC 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).
Results:
PC-3M cells showed higher hsa-miR-4516 expression and higher PrPC protein levels than LNCaP-LN3 cells. In PC-3M cells, miR-4516 inhibition reduced PRNP mRNA and PrPC protein levels, decreased CDK4, cyclin D1, and cyclin E expression, and reduced cell numbers. Milk EV-siPRNP decreased PrPC 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.
Conclusion:
In PC-3M cells, inhibition of hsa-miR-4516 or PRNP knockdown reduced PRNP/PrPC 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.
