Elsevier

Urology

Volume 91, May 2016, Pages 241.e1-241.e7
Urology

Basic and Translational Science
PLK-1 Silencing in Bladder Cancer by siRNA Delivered With Exosomes

https://doi.org/10.1016/j.urology.2016.01.028Get rights and content

Objective

To use exosomes as a vector to deliver small interfering ribonucleic acid (siRNA) to silence the polo-like kinase 1 (PLK-1) gene in bladder cancer cells.

Materials and Methods

Exosomes were isolated from both human embryonic kidney 293 (HEK293) cell and mesenchymal stem cell (MSC) conditioned media. Fluorescently labeled exosomes were co-cultured with bladder cancer and normal epithelial cells and uptake was quantified by image cytometry. PLK-1 siRNA and negative control siRNA were loaded into HEK293 and MSC exosomes using electroporation. An invasive bladder cancer cell line (UMUC3) was co-cultured with the electroporated exosomes. Quantitative reverse transcriptase polymerase chain reaction was performed. Protein analysis was performed by Western blot. Annexin V staining and MTT assays were used to investigate effects on apoptosis and viability.

Results

Bladder cancer cell lines internalize an increased percentage of HEK293 exosomes when compared to normal bladder epithelial cells. Treatment of UMUC3 cells with exosomes electroporated with PLK-1 siRNA achieved successful knockdown of PLK-1 mRNA and protein when compared to cells treated with negative control exosomes.

Conclusion

HEK293 and MSC exosomes were effectively used as a delivery vector to transport PLK-1 siRNA to bladder cancer cells in vitro, resulting in selective gene silencing of PLK-1. The use of exosomes as a delivery vector for potential intravesical therapy is attractive.

Section snippets

Cell Culture

UMUC3, SW780, and HEK293 cell lines were purchased from ATCC and cultured in Dulbecco's modified eagle medium containing 10% fetal bovine serum, 100 units/mL penicillin, 100 µg/mL streptomycin, and 2 mmol/L L-glutamine. Normal urothelial cells were purchased from Zen-Bio (Research Triangle Park, NC) and cultured in CnT-Prime Epithelial Culture Medium (Zen-Bio). Cells tested negative for mycoplasma contamination using the MycoAlert Plus Mycoplasma detection kit (Lonza) and they were not cultured

Internalization of HEK293 Exosomes by Bladder Cancer Cells and Normal Urothelial Cells

With the idea of ultimately using exosomes as an intravesical therapy, we investigated the rate of uptake of HEK293 exosomes in bladder cancer cells vs normal urothelial cells. PKH-26-labeled HEK293 exosomes were analyzed on the ImageStreamX after incubating for 6 hours with bladder cells lines UMUC3 and SW780, or with normal urothelial cells. We found that the bladder cancer cells internalize HEK293 exosomes more avidly than normal urothelial cells. Both bladder cancer cell lines tested took

Discussion

Genetic therapeutics have been limited by the availability of an efficient delivery vector. The delivery of siRNA into a cell's cytosol is challenging because it is rapidly degraded, and their size and negative charge limit cellular uptake.13 In addition, siRNA can elicit a powerful immune response, especially with repeated administration. The encapsulation of nucleic acid therapeutics into delivery vectors is a promising strategy to overcome these challenges.13 The ideal delivery vector has

Conclusion

Exosomes have many natural properties that make them an ideal delivery vector for genetic therapeutics. This study adds to the growing body of literature showing the successful use of exosomes as delivery vectors, and it is the first study evaluating their use in bladder cancer with implications in the intravesical therapy setting. Future directions will include alternative gene targets and in vivo models.

References (15)

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Financial Disclosure: The authors declare that they have no relevant financial interests.

Funding Support: This work was supported in part by the 2013 Urology Care Foundation Residency Research Award to Kristin A. Greco.

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