Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcription profiling of mouse mast cells derived exosomal RNA versus their parental cells MC/9.

ABSTRACT: Exosomes are vesicles of endocytic origin released by many types of cells into the extracellular environment. In an attempt to further examine the exosome-mediated cellular communication, we show that exosomes from a mouse mast cell line (MC/9), exosomes from primary bone marrow derived mast cells, and exosomes from a human mast cell line (HMC-1) contain RNA but not DNA. Microarray assessments of exosome-derived RNA revealed that these vesicles contain mRNA from approximately 1200 genes, many of which are unique and not present in the cytoplasmic RNA pool in the donor cell. Experiment Overall Design: Exosomes were prepared from the supernatant of MC/9 cells by differential centrifugations and filtration. RNA was isolated from the exosomes and their parental cells using Trizo. The microarray experiments were performed by SweGene (www.swegene.org/) according to Affymetrix microarray DNA chip analysis. The experiment was performed in quadruple samples. ExoRNA1, ExoRNA2, ExoRNA3, and ExoRNA4 for the exosomes samples and Mast_cells1, Mast_cells2, Mast_cells3, and Mast_cells4 for the MC/9 cells.

ORGANISM(S): Mus musculus

SUBMITTER: Hadi Valadi

PROVIDER: E-GEOD-7275 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.

Valadi Hadi H Ekström Karin K Bossios Apostolos A Sjöstrand Margareta M Lee James J JJ Lötvall Jan O JO

Nature cell biology 20070507 6

Exosomes are vesicles of endocytic origin released by many cells. These vesicles can mediate communication between cells, facilitating processes such as antigen presentation. Here, we show that exosomes from a mouse and a human mast cell line (MC/9 and HMC-1, respectively), as well as primary bone marrow-derived mouse mast cells, contain RNA. Microarray assessments revealed the presence of mRNA from approximately 1300 genes, many of which are not present in the cytoplasm of the donor cell. In vi ...[more]

PMID: 17486113

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Project description:Background: Exosomes are nanovesicles of endocytic origin believed to be involved in communication between cells. Recently, it has been shown that mast cell exosomes contain RNA named "exosomal shuttle RNA". The aim of this study was to evaluate whether exosomal shuttle RNA could play a role in the communication between human mast cells and between human mast cells and human CD34 positive progenitor cells. Results: Exosomes from the human mast cell line HMC-1 contain RNA. The exosomes contain no or very little ribosomal RNA compared to their donor cells. The mRNA and microRNA content in exosomes and their donor cells was examined using microarray analyses. We found 116 microRNA in the exosomes and 134 microRNA in the cells, from which some were expressed at different level. DNA microarray experiments revealed the presence of approximately 1800 mRNAs in the exosomes, which represent 15% of the donor cell mRNA content. Transfer experiments revealed that exosomes and their RNA can transfer to other HMC-1 cells and to CD34 positive progenitors. Conclusions: To conclude, HMC-1 exosomes contain mRNA and microRNA that can be transferred to other mast cells and to CD34 progenitors. This shuttle of exosomal RNA may represent a powerful mode of communication between cells where cells send genetic information to other cells over a distance via exosomes. [miRNA profiling] Identification of microRNA was performed by Exiqon (www.exiqon.com). Briefly, the quality of the total RNA was verified by an Agilent 2100 Bioanalyzer. Total RNA from the exosome and the HMC-1 cell samples were labelled with Hy3 and Hy5 fluorescent stain, respectively, using the miRCURY Hy3/Hy5 power labelling kit. The Hy3-labelled exosome samples and a Hy5-labeled mast cells were mixed pair-wise and hybridized to the miRCURY? LNA array (v9.2). The hybridization was performed according to the miRCURY? LNA array manual using a Tecan HS4800 hybridization station (Tecan Systems, Inc. San Jose, CA). The miRCURY? LNA array microarray slides were scanned by a ScanArray 4000 XL scanner (Packard Biochip Technologies, Billerica, MA ,USA) and the image analysis was carried out using the ImaGene 6.1.0 software (BioDiscovery, Inc, El Segundo, CA USA). The quantified signals were normalized using the global Lowess (LOcally WEighted Scatterplot Smoothing) regression algorithm. MicroRNA with signals equal to or below the background signal in 2 or more of the 4 replicate measurements were identified as absent in that slide. The limit for a miRNA to be listed as detectable was set to signal intensities higher than 3 x background (3 x median Hy3 or Hy5 for the total slide). In addition, where signals were detected for <3 of the slides, they were considered unreliable and excluded from sets of detected miRNAs. The experiment was performed in triplicate samples. The signal was calculated as the mean value of the log2MeanRatio Hy3/Hy5 for the triplicates M-BM-1 SD. [mRNA profiling] Exosomes were prepared from the supernatant of HMC-1 cells by differential centrifugations and filtration. RNA was isolated from the exosomes and their parental cells using Trizol . The microarray experiments were performed by SweGene (www.swegene.org/) according to Affymetrix microarray DNA chip analysis (n=4). p0739_E1, p0739_ E2, p0739_E3 and p0739_E4 for the exosomes samples and p0739_C1, p0739_C2, p0739_C3, and p0739_C4 for the HMC-1 cells. [miRNA profiling] Exosomes were prepared from the supernatant of HMC-1 cells by differential centrifugations and filtration. RNA was isolated from the exosomes and their parental cells using Trizol followed by RNeasy clean-up. The microarray experiments were performed by Exiqon.

Project description:Background: Docosahexaenoic acid (DHA) is a natural compound with anticancer and anti-angiogenesis activity that is currently under investigation as both a preventative agent and an adjuvant to breast cancer therapy. However, the precise mechanisms of DHA’s anticancer activities are unclear. It is understood that the intercommunication between cancer cells and their microenvironment is essential to tumor angiogenesis. Exosomes are extracellular vesicles that are important mediators of intercellular communication and play a role in promoting angiogenesis. However, very little is known about the contribution of breast cancer exosomes to tumor angiogenesis or whether exosomes can mediate DHA’s anticancer action. Results: Exosomes were collected from MCF7 and MDA-MB-231 breast cancer cells after treatment with DHA. We observed an increase in exosome secretion and exosome microRNA contents from the DHA-treated cells. The expression of 83 microRNAs in the MCF7 exosomes was altered by DHA (>2-fold). The most abundant exosome microRNAs (let-7a, miR-23b, miR-27a/b, miR-21, let-7, and miR-320b) are known to have anti-cancer and/or anti-angiogenic activity. These microRNAs were also increased by DHA treatment in the exosomes from other breast cancer lines (MDA-MB-231, ZR751 and BT20), but not in exosomes from normal breast cells (MCF10A). When DHA-treated MCF7 cells were co-cultured with or their exosomes were directly applied to endothelial cell cultures, we observed an increase in the expression of these microRNAs in the endothelial cells. Furthermore, overexpression of miR-23b and miR-320b in endothelial cells decreased the expression of their pro-angiogenic target genes (PLAU, AMOTL1, NRP1 and ETS2) and significantly inhibited tube formation by endothelial cells, suggesting that the microRNAs transferred by exosomes mediate DHA’s anti-angiogenic action. These effects could be reversed by knockdown of the Rab GTPase, Rab27A, which controls exosome release. Conclusions: We conclude that DHA alters breast cancer exosome secretion and microRNA contents, which leads to the inhibition of angiogenesis. Our data demonstrate that breast cancer exosome signaling can be targeted to inhibit tumor angiogenesis and provide new insight into DHA’s anticancer action, further supporting its use in cancer therapy. Examination of small RNA populations in MCF7 cells and exosomes after DHA treatment.

Dataset Information

Transcription profiling of mouse mast cells derived exosomal RNA versus their parental cells MC/9.

Publications

Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.

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