Project description:Exosomes are molecular entities derived from membrane vesicles of endocytic origin secreted by most cell types. These vesicles are implicated in cell-to-cell communication, deliver proteins and mRNA molecules between cells. Recent studies have shown that exosomes are found in body fluids such as saliva, blood, urine, amniotic fluid, malignant ascites, bronchoalveolar lavage fluid, synovial fluids and breast milk. Exosomes secreted through human saliva contain mRNA may potentially be useful for diagnostic purposes. Although the exact protective mechanism of saliva RNA is a topic of debate, the consensus is that the enrichment of mRNAs in these nano-vesicles in one of the features of the biomarker discoveries. Our aim was to determine if exosomes are present in human saliva and to nano-characterize their transcriptomic content. Exosomes were purified by differential ultracentrifugation, identified by immunoelectron microscopy, flow cytometry and western blot using a CD-63 antibody. Atomic force microscopy studies revealed ultra structural analysis of both size and density of exosomes. Microarray analysis revealed the presence of 590 mRNA core transcripts are relatively stable inside the exosomes, which can be of saliva mRNA biomarkers. Exosomal mRNA stability was determined by detergent lyses with treatment of RNase. Under in vitro conditions fluorescent dye labeled saliva exosomes were able to communicate between human oral keratinocytes studied by using fluorescence microscopy. The RNA from saliva exosomes can transfer their genetic information to human oral keratinocytes and alters gene expression in the new location. Together, these results suggest that saliva is involved in mRNA trafficking via exosomes, and provides a mechanism for cargoing passenger mRNAs. Our findings are consistent with proposal that exosomes can shuttle RNAs between cells and mRNA is protected inside these vesicles may be a possible resource for biomarker discovery. Experiment Overall Design: Human saliva exosomes were purified through differential centrifugation followed by RNA extraction and hybridization on Affymetrix microarrays. We were able to obtain normal human subjects saliva which are pooled and subjected to ultracentrifugation. The protocol was approved by UCLA Institutional review board. 1 ml of saliva exosomes were used to extract RNA followed by two rounds of amplification by Actorus Amp kit. The amplified RNA was biotin labled and hybridized with Affymetrix protocol.

Project description:Exosomes are molecular entities derived from membrane vesicles of endocytic origin secreted by most cell types. These vesicles are implicated in cell-to-cell communication, deliver proteins and mRNA molecules between cells. Recent studies have shown that exosomes are found in body fluids such as saliva, blood, urine, amniotic fluid, malignant ascites, bronchoalveolar lavage fluid, synovial fluids and breast milk. Exosomes secreted through human saliva contain mRNA may potentially be useful for diagnostic purposes. Although the exact protective mechanism of saliva RNA is a topic of debate, the consensus is that the enrichment of mRNAs in these nano-vesicles in one of the features of the biomarker discoveries. Our aim was to determine if exosomes are present in human saliva and to nano-characterize their transcriptomic content. Exosomes were purified by differential ultracentrifugation, identified by immunoelectron microscopy, flow cytometry and western blot using a CD-63 antibody. Atomic force microscopy studies revealed ultra structural analysis of both size and density of exosomes. Microarray analysis revealed the presence of 590 mRNA core transcripts are relatively stable inside the exosomes, which can be of saliva mRNA biomarkers. Exosomal mRNA stability was determined by detergent lyses with treatment of RNase. Under in vitro conditions fluorescent dye labeled saliva exosomes were able to communicate between human oral keratinocytes studied by using fluorescence microscopy. The RNA from saliva exosomes can transfer their genetic information to human oral keratinocytes and alters gene expression in the new location. Together, these results suggest that saliva is involved in mRNA trafficking via exosomes, and provides a mechanism for cargoing passenger mRNAs. Our findings are consistent with proposal that exosomes can shuttle RNAs between cells and mRNA is protected inside these vesicles may be a possible resource for biomarker discovery. Keywords: Human saliva, exosomes, mRNA profiling, gene expression, disease diagnosis

Project description:Exososmes, potent intercellular communicators, are supposed to contribute to metastasis formation, which we confirmed for exosomes of the metastatic rat pancreatic adenocarcinoma line BSp73ASML that promote metastatic settlement in lymph nodes and lung of poorly metastatic BSp73ASML cells with a selective CD44v4-v7 (BSp73ASML-CD44vkd) knockdown. To define the molecular pathway(s), whereby exosomes contribute to premetastatic niche preparation, we profiled mRNA miRNA of BSp73ASMLwt and BSp73ASML-CD44vkd- exosomes and evaluated the impact on potential target cells. BSp73ASML exosomes are recovered in the draining lymph node after subcutaneous injection. In vitro, they preferentially bind and are taken-up by lymph node stroma cells (LnStr) and lung fibroblasts (LuFb) that were chosen as exosome targets. BSp73ASMLwt and BSp73ASML-CD44kd exosomes contain a restricted repertoire of mRNA and miRNA, hwere the lattter differe significantly between the two lines and even more pronounced, exosomes derived thereof with a not yet explored dominance of tumor-suppressor miRNA in ASML-CD44kd cells and exosomes. Both, exosomal mRNA and miRNA are recovered in target cells and exosome-uptake is accompanied by significant changes in gene expression. We didn't observe a correlation between exosomal mRNA and changes in target cell mRNA or proteins. Instead transferred miRNA significantly affected target cell mRNA translation as demonstrated for selected, most abundant ASML exosomal miRNA besides others, miR-494 known target MAL (myelin and lymphocytes protein)/cadherin17, and miR-542-3p which targets TRAF/cadherin17. Furthermore, MMP transcription suggested to accompany cadherin17 dwon-regulation was upregulated in miR-494 or miR542-3p transfected or exosome co-cultured LnStr. Taken together, tumor exosomes target in vivo non-transformed cells in premetastatic organs. Exosome uptake induced altered target celll gene expression is strongly promoted by exosomal miRNA where we demonstrate for the first time that exosomes/exosomal miRNA from a metastasizing tumor line can modulate stroma cells from premetastatic organs. Endothelial cells lines were treated with pancreatic adenocarcinoma (AS) derived exosomes or pancreatic adenocarcinoma derived exosomes expressing tetraspanin 8. Total RNA was isolated and used to perform the Agilent gene expression microarrays. In this assay a replicate of endothelial cell lines treated with ASTspan8 were also included. Moreover, total RNA from both base line expression of endothelial cells and rat endothelial fibroblasts were also used to perfrom gene expression microarrays. RNA isolated from Rat endothelial fibroblasts treated with the exosomes derived from rat pancreatic adenocarcinoma and exosomes derived from rat pancreatic adenocarcinoma expressing tetraspanin8 were individually used to perfrom gene expression microarrays. RNA isolated from exosomes derived from rat pancreatic adenocarcinoma cell lines expressing tetraspanin were used to peform gene expresiion to see the base line expression. Another replicate were also used. RNA isolated from base line or control of rat pancreatic adenocarcinoma wild type cells and also base line RNA isolated from rat pancreatic adenocarcinoma cells lines where CD44 was knock-down.

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Exosomal miRNA profiling of saliva-derived exosomes

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