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.

Figure 1. EM image of human saliva showing round-shaped exosomes.

The 120,000×g pellets from saliva were used for exosomes analysis. (A) Electron micrographs of saliva exosomes were fixed in 2% formaldehyde and contrasted using 2% uranyl acetate. The image shows small vesicles of ∼60 nm in diameter. (B) Exosomes were labeled with immunogold anti-CD63. Note the immunoreactivity of CD63 on the surface of the single exosome. (C) Representative FACS analysis of exosomes showing expression of CD63. Open trace shows the saliva exosomes, filled trace shows saliva exosomes incubated with latex beads and stained with anti-CD63 followed by secondary Alexa Fluor 488-conjugated antibody. (D) Western blot analysis of saliva exosomes using an antibody against CD63 and Alix. Lane 1 is the protein extract of normal saliva, and lane 2 contains protein from the exosome pellet obtained from ultracentrifugation.

Figure 2. AFM images of saliva exosomes.

Exosomes (panels B–F) were adsorbed to WGA-coated mica surfaces. (A) Topography images were obtained with the use of the Mac mode in water (negative control—no exosomes). (B) A 3D AFM image of isolated exosomes adhering to a mica sheet. The bar denotes 200µM. (C) A high-resolution single image of the exosome structure on the mica. (D) Graphical representation of height and width of a single exosome. (E) Size distribution of several saliva exosomes imaged with AFM. (F) Graphical representation of the size distribution of exosomes showing near homogeneity with respect to height and width.

Figure 3. Exosomes contain mRNA species.

(A) RNA from saliva exosomes was detected using an Agilent bioanalyser. Lane 1, RNA ladder showing sizes of the nucleotides on the left. Representative lanes (2–5) showing sizes of the mRNA species identified using an Agilent bioanalyser electrophorogram. The saliva exosomal RNA contains no ribosomal RNA as seen by the small heterogeneous RNA fragments (<200 nucleotides). (B) Bioanalyzer graphical data shows the size distribution of total RNA extracted from saliva exosomes (1) profile of RNA standard (2) total RNA extracted from saliva exosomes without any treatments (3) total RNA treated with DNase (4) total RNA treated with RNase A and (5) total RNA treated with both DNase and RNase A. (C) The biological process ontology of the 509 core mRNA species identified in the saliva exosomes.

Figure 4. Saliva exosomes treated with Triton X-100 and RNase had different RNA content compared to control, indicating that RNA is protected inside the exosomes.

Higher Ct values represent lower RNA content. Error bars denote SEM (n = 3).

Figure 5. Oral keratinocytes (5×10⁷ cells/well) were incubated for 24 hr in KSFM media with fluorescently labeled exosomes and examined under fluorescence microscopy.

The lysed lanes serve as a negative control. Magnification was 10×, and the smaller boxed panels represent magnification of 40×. Note the fluorescence intensity increases with increasing amounts of exosomes (32 and 64 µl, respectively).

Figure 6. Identification of differentially expressed proteins from oral keratinocytes treated with saliva exosomes by 2-DIGE.

Proteins from cells treated with the negative control were labeled with Cy3 (green) and proteins from cells treated with saliva exosomes were labeled with Cy5 (red). Isoelectric focusing was carried out at pH 3–10, and 2D separation was performed with 8–14% gradient SDS-PAGE. The negative control represents protein profiles of keratinocytes treated with detergent lysed exosomes. The bottom gel image reveals differentially expressed proteins in the control and treated samples after merging. Protein spots shown in red are presumably due to upregulation by exosome treatment, and those in green are due to downregulation by exosome treatment. Such spots are circled and numbered.

Figure 7. Protein expression modulated in saliva exosomes.

ANXA1, ANXA2, moesin, keratin-6A, EEF2, OS-9, and IL-8 RNA expression was normalized using a factor calculated from β-Actin gene expression.