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:Recent evidence suggests that hypoxia caused by acute myocardial infarction (AMI) can induce evident cardiomyocyte apoptosis. Exosomes, as paracrine signaling mediators by delivering cytosolic components (including miRNA, mRNA, protein), play crucial roles in intercellular communication. However, the systemic regulation of exosome-mediated miRNA delivery and subsequent regulation effect underlying AMI are not well-understood to date. Here, using small RNA-seq, we investigated the miRNAome of H9C2 cells and corresponding paracrine exosmes after hypoxia stress, and identified total of 92 and 62 differentially expressed (DE) miRNAs in cells and exosomes from hypoxia vs. normoxia condition. We found that hypoxia strongly induced the expression of hypoxamiRs in H9C2 cells and altered cardiomyocyte-derived exosomal miRNAome. Functional enrichment analysis revealed extensive roles of DE exosomal miRNAs in HIF-1 signaling pathway and cell apoptosis process, such as TNF signaling pathway, MAPK signaling pathway, mTOR signaling pathway. Furthermore, the gain- and loss-of-function analysis demonstrated the anti-apoptotic effects of hypoxia-induced exosomal miRNAs, i.e. miR-21-5p, miR-378-3p, miR-152-3p, let-7i-5p, and then the luciferase reporter assay confirmed that miR-152-3p and let-7i-5p implemented their anti-apoptotic function by directly targeting Atg12 and Faslg, respectively. In short, this study revealed that hypoxia-exosomes derived from cardiomyocyte, loaded abundant cardio-protective miRNAs, mediate crosstalk among cardiomyocytes and prevent cardiomyocytes apoptosis after hypoxia. Methods: we established the anoxia model using H9C2 cells, an immortal cardiac muscle cell line, to imitate hypoxia condition caused by AMI in vitro, and then small RNA-seq were employed to investigate the miRNA transcriptome of H9C2 cells and its exosomes collected from hypoxia and normoxia culture medium. Results: we identified the miRNAome of H9C2 cells and its exosmes under both hypoxia and normoxia, including 331, 338, 144, 74 unique mature miRNAs from hypoxia-cells, normoxia-cells, hypoxia-exosomes, normoxia-exosomes, respectively. Total of 92 and 62 DE miRNAs were identified from cells and exosomes. Among of which, the DE exosomal miRNAs were mainly involved in HIF-1 signaling pathway and cell apoptosis process, such as TNF signaling pathway, MAPK signaling pathway, mTOR signaling pathway. Interestingly, we found that some DE exosomal miRNAs, including miR-21-5p, miR-378-3p, miR-152-3p, let-7i-5p, have an anti-apoptotic and pro-viability effects in H9C2 cells under hypoxic stress. In addition, Atg12 and Faslg to be respective target of miR-152-3p and let-7i-5p were partly elucidated the anti-apoptosis mechanism of hypoxia-exosomes. Conclusions:In brief, this study illustrated a potential mechanism that, under hypoxic stress, hypoxia pre-perceived cardiomyocytes can spontaneously secrete the hypoxamiRs enriched exosomes, loaded a large amount of cardio-protective miRNA, which mediate crosstalk among cardiomyocytes and prevent apoptosis to heighten the hypoxia adaptability of cardiomyocytes. In more detail, we first discovered that miR-152-3p and let-7i-5p, enriched in hypoxia-exosomes derived by cardiomyocytes, played an anti-apoptosis role via mitochondrial pathway(intrinsic pathway) and death receptor pathway (extrinsic pathway), respectively.

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.

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