Kristin A Greco1, Carrie A Franzen1, Kimberly E Foreman2, Robert C Flanigan1, Paul C Kuo3, Gopal N Gupta4. 1. Department of Urology, Loyola University Chicago, Maywood, IL. 2. Department of Pathology, Loyola University Chicago, Maywood, IL; Oncology Institute, Loyola University Chicago, Maywood, IL. 3. Oncology Institute, Loyola University Chicago, Maywood, IL; Department of Surgery, Loyola University Chicago, Maywood, IL. 4. Department of Urology, Loyola University Chicago, Maywood, IL; Oncology Institute, Loyola University Chicago, Maywood, IL; Department of Surgery, Loyola University Chicago, Maywood, IL; Department of Radiology, Loyola University Chicago, Maywood, IL. Electronic address: gogupta@lumc.edu.
Abstract
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.
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 humanembryonic 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.
Authors: Andrew E Massey; Shabnam Malik; Mohammad Sikander; Kyle A Doxtater; Manish K Tripathi; Sheema Khan; Murali M Yallapu; Meena Jaggi; Subhash C Chauhan; Bilal B Hafeez Journal: Int J Mol Sci Date: 2021-05-17 Impact factor: 5.923