Plasma exosome from mice bearing Ago2wt and Ago2dm tumor
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ABSTRACT:
ORGANISM(S): synthetic construct Mus
PROVIDER: GSE239599 | GEO | 2024/04/18
REPOSITORIES: GEO
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Project description:Type 1 diabetes mellitus (T1DM) results from an autoimmune attack against the insulin-producing ß cells which leads to chronic hyperglycemia. Exosomes are lipid vesicles derived from cellular multivesicular bodies that are enriched in specific miRNAs, potentially providing a disease-specific diagnostic signature. To assess the value of exosome miRNAs as biomarkers for T1DM, miRNA expression in plasma-derived exosomes was measured. Nanoparticle tracking analysis and transmission electron microscopy confirmed the presence of plasma-derived exosomes (EXOs) isolated by differential centrifugation. Total RNA extracted from plasma-derived EXOs of 12 T1DM and 12 control subjects was hybridized onto Nanostring human v2 miRNA microarray array and expression data were analyzed on nSolver analysis software. We found 7 different miRNAs (1 up-regulated and 6 down-regulated), that were differentially expressed in T1DM. The selected candidate miRNAs were validated by qRT-PCR analysis of cohorts of 24 T1DM and 24 control subjects. Most of the deregulated miRNAs are involved in progression of T1DM. These findings highlight the potential of EXOs miRNA profiling in the diagnosis as well as new insights into the molecular mechanisms involved in T1DM.
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2014-06-14 | E-GEOD-58464 | biostudies-arrayexpress
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.
2008-11-15 | E-GEOD-13494 | biostudies-arrayexpress
Project description:PURPOSE: To investigate the circulatory microRNA (miRNA) profiles of aqueous, vitreous, and plasma in order to identify biomarkers in aqueous humor or plasma that are reflecting changes in vitreous of patients with diabetes. METHODS: Aqueous, vitreous and plasma samples were collected from a total of 27 patients - 11 controls (macular pucker or macular hole patients) and 16 patients with diabetes mellitus (DM) undergoing vitreoretinal surgery: DM-Type I with proliferative diabetic retinopathy (PDR) (DMI-PDR), DM Type II with PDR (DMII-PDR) and DM Type II with nonproliferative DR (DMII-NPDR). MiRNAs were isolated using Qiagen microRNeasy kit, quantified on BioAnalyzer, labeled with FlashTag kit, and profiled on Affymetrix GeneChip miRNA 3.0 microarrays. Data analysis was done using Expression Console (EC), Transcriptome Analysis Console (TAC), and Ingenuity Pathway Analysis (IPA) software. RESULTS: Our comparison of circulatory miRNA population of aqueous and vitreous humor and plasma showed that out of total of 847 human miRNA probes on the Affymetrix GeneChip miRNA 3.0 we found common miRNAs for both aqueous and vitreous samples, as well as larger number of unique miRNA, dependent on the DM type and presence of retinopathy. Most of the dysregulated miRNAs in aqueous and vitreous of DM patients were upregulated, while in plasma, most of the DM-specific miRNAs were downregulated. Dysregulation of miRNAs in aqueous generally do not appear to be a good representative of the miRNA abundance in vitreous, or plasma, although we did identify a few candidates for common biomarkers: let-7b, miR-320b, miR-762 and miR-4488. Additionally, each of the DR subtypes showed a set of miRNA that is uniquely dysregulated in each fluid, for example in aqueous samples for DMII-NPDR it was miR-455-3p, for DMII-PDR was miR-296, and for DMI-PDR it was miR-3202. Pathway analysis identified TGF-beta and VEGF pathways as the common targets for miRNAs dysregulated in DR aqueous and vitreous. CONCLUSIONS: The comparative profiling of circulatory miRNAs in aqueous, vitreous, and plasma showed that a small number of circulatory miRNAs displayed differential presence in controls vs. diabetic retinopathy. A pattern is emerging of sets of miRNA that are common or uniquely dysregulated in the blood plasma or ocular fluids of DR subtypes, offering promise for the use of ocular fluids and plasma for identifying diagnostic and therapeutic targets.
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Project description:Introduction: microRNAs are promising candidate breast cancer biomarkers due to their cancer-specific expression profiles. However, efforts to develop circulating breast cancer biomarkers are challenged by the heterogeneity of microRNAs in the blood. To overcome this challenge, we aimed to develop a molecular profile of microRNAs specifically secreted from breast cancer cells. Our first step towards this direction relates to capturing and analyzing the contents of exosomes, which are small secretory vesicles that selectively encapsulate microRNAs indicative of their cell of origin. To our knowledge, circulating exosome microRNAs have not been well evaluated as biomarkers for breast cancer diagnosis or monitoring. Methods: Exosomes were collected from the conditioned media of human breast cancer cell lines, mouse plasma of patient-derived orthotopic xenograft models (PDX), and human plasma samples. Exosomes were verified by electron microscopy, nanoparticle tracking analysis, and western blot. Cellular and exosome microRNAs from breast cancer cell lines were profiled by next-generation small RNA sequencing. Plasma exosome microRNA expression was analyzed by qRT-PCR analysis. Results: Small RNA sequencing and qRT-PCR analysis showed that several microRNAs are selectively encapsulated or highly enriched in breast cancer exosomes. Importantly, the selectively enriched exosome microRNA, human miR-1246, was detected at significantly higher levels in exosomes isolated from PDX mouse plasma, indicating that tumor exosome microRNAs are released into the circulation and can serve as plasma biomarkers for breast cancer. This observation was extended to human plasma samples where miR-1246 and miR-21 were detected at significantly higher levels in the plasma exosomes of 16 breast cancer patients as compared to the plasma exosomes of healthy control subjects. Receiver Operating Characteristic (ROC) curve analysis indicated that the combination of plasma exosome miR-1246 and miR-21 levels is a better indicator of breast cancer than their individual levels. Conclusions: Our results demonstrate that certain microRNA species, such as miR-21 and miR-1246, are selectively enriched in human breast cancer exosomes and significantly elevated in the plasma of breast cancer patients. These findings indicate a potential new strategy to selectively analyze plasma breast cancer microRNAs indicative of the presence of breast cancer.
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