Abstract
Glioblastoma is a very invasive and prevalent brain tumor that affects 15 in 100,000 persons over the age of 70 years. Studies have shown that the expression of the WD repeat domain 81 (WDR81) gene, which is effective in vesicular transport and inhibition of autophagy, is increased in glioblastoma. The decreased autophagy was found to be related to the increased production of exosomes, which is a major factor in the pathogenesis of glioblastoma. The PI-3kinase complex is a pre-autophagic complex that is highly active in the absence of WDR81. The WDR81 gene, as a negative regulator of PI3K activity, prevents autophagy and increases exosome secretion by preventing the formation of the class III PI3K complex. Therefore, targeted reduction of exosomes can be considered an effective strategy for reducing the pathogenesis of glioblastoma. This study aimed to assess the effect of WDR81 gene silencing with siRNA on exosome levels in a U87-MG cell line. Culturing of U87-MG cells was carried out in Dulbecco’s modified Eagle medium (DMEM) containing 5% FBS and 1% penicillin/streptomycin. Thereafter, silencing of WDR81 was performed using WDR81 siRNA, whose gene expression level was determined via real-time qRT-PCR. Cell viability was evaluated using the MTT assay. The exosomes were extracted from a cell culture using the Exocib kit. The size accuracy of the exosomes was confirmed by dynamic light scattering (DLS). Finally, the protein content and RNA of the exosomes were assessed. WDR81 gene expression of siRNA-transfected cells was decreased to 82% after 24 h compared to the non-transfected control cells. The analysis of the exosomes showed that the concentration of exosomes and their RNA and protein content in the siRNA-transfected cells decreased significantly compared to the non-transfected control cells. No considerable difference was observed in cell viability after transfection with either WDR81-specific siRNAs or scrambled control siRNAs. Our findings showed that silencing the WDR81 gene could reduce the level of exosomes in human U87-MG glioblastoma cells. Therefore, the reduced exosome content may be suggested as a new gene therapy strategy for targeted therapy of glioblastoma by increasing autophagy via activation of PI3KIII. However, more studies are needed in this regard.
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Data Availability Statement
The data supporting the findings of this study are available from the corresponding author upon reasonable request.
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Acknowledgments
The authors would like to thank the professors and staff of the Faculty of Advanced Medical Technologies, Golestan University of Medical Sciences, Gorgan, Iran.
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This study was supported by the Stem Cell Research Center, Golestan University of Medical Sciences (research grant number 111026).
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ZB, AK, and AT jointly designed the study and experiments. Material preparation, experiments, and data analysis were performed by AT. ZB and AK gave advice on performing the experiments. The whole study was done under the supervision of MO. AT wrote the first draft of the manuscript. MO edited and revised the manuscript. All authors read and approved the final manuscript.
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Tadayoni nia, A., Bazi, Z., Khosravi, A. et al. WDR81 Gene Silencing Can Reduce Exosome Levels in Human U87-MG Glioblastoma Cells. J Mol Neurosci 71, 1696–1702 (2021). https://doi.org/10.1007/s12031-021-01849-z
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DOI: https://doi.org/10.1007/s12031-021-01849-z