Nanostructural and Transcriptomic Analyses of Human Saliva Derived Exosomes

Abstract

BACKGROUND: Exosomes, derived from endocytic membrane vesicles are thought to participate in cell-cell communication and protein and RNA delivery. They are ubiquitous in most body fluids (breast milk, saliva, blood, urine, malignant ascites, amniotic, bronchoalveolar lavage, and synovial fluids). In particular, exosomes secreted in human saliva contain proteins and nucleic acids that could be exploited for diagnostic purposes. To investigate this potential use, we isolated exosomes from human saliva and characterized their structural and transcriptome contents. METHODOLOGY: Exosomes were purified by differential ultracentrifugation and identified by immunoelectron microscopy (EM), flow cytometry, and Western blot with CD63 and Alix antibodies. We then described the morphology, shape, size distribution, and density using atomic force microscopy (AFM). Microarray analysis revealed that 509 mRNA core transcripts are relatively stable and present in the exosomes. Exosomal mRNA stability was determined by detergent lysis with RNase A treatment. In vitro, fluorescently labeled saliva exosomes could communicate with human keratinocytes, transferring their genetic information to human oral keratinocytes to alter gene expression at a new location. CONCLUSION: Our findings are consistent with the hypothesis that exosomes shuttle RNA between cells and that the RNAs present in the exosomes may be a possible resource for disease diagnostics.