The expression profiles of miRNAs in exosomes derived from H2O2-treated and untreated HaCaT cells
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PROVIDER: PRJNA1029895 | ENA |
REPOSITORIES: ENA
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Project description:Vitiligo is a skin disease characterized by the destruction of epidermal melanocytes due to oxidative stress and autoimmune response. As the main responder to oxidative stress, keratinocytes facilitate melanocyte loss under oxidative stress by inducing melanocyte death and recruiting antigen-specific CD8+ T cell to skin to destroy melanocytes. It has been proved that keratinocytes secrete abundant functional exosomes, but the role of exosomes secreted from keratinocytes under oxidative stress in vitiligo pathogenesis is unknown. We found that oxidative stress enhanced the secretion of exosomes from keratinocytes. These exosomes (OS-Exos) administration aggravated melanocyte loss and CD8+ T cell infiltration in the epidermis of tail in the vitiligo mouse model, thereby driving vitiligo progression. OS-Exos suppressed the survival of melanocytes while promoting the proliferation and activation of CD8+ T cells in vitro. As miRNAs contained in OS-Exos might be responsible for the functions of OS-Exos on melanocytes and CD8+ T cells, we employed small RNAs-seq analysis to screen miRNAs enriched in OS-Exos as compared to those in Exos (from untreated HaCaT cells). The hierarchical clustering analysis and the volcano plot showed the distinct signatures of known miRNAs as well as novel miRNAs between OS-Exos and Exos. In total, 276 differentially expressed miRNAs were discovered, with 134 and 142 miRNAs displaying significant up- or down-regulation, respectively, in OS-Exos compared with Exos (P<0.05; fold change≥2. The above data suggest that miRNAs enriched in OS-Exos might contribute to the facilitatory role of OS-Exos in vitiligo progression.
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Project description:H2O2-treated and untreated HCT116 cells
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Project description:ChIP-on-chip tilling array comparing untreated human SW480 cells vs SW480 cells treated with 2mM H2O2 for 30min. Exposing cells to the reactive oxygen species, hydrogen peroxide, recruits DNA methyltransferase 1 (DNMT1) to damaged chromatin. DNMT1 becomes part of a complex(es) containing DNMT3B and members of Polycomb Repressive Complex 4. The goal was to determine the effect of hydrogen peroxide treatment on chromatin, including changes in histone modifications and binding patterns of chromatin-associated proteins. [Agilent-014706]: Two-condition: untreated SW480 cells (U) vs H2O2 treated SW480 cells (T). Five-mark: SIRT1, gamma-HA2X, DNMT1, DNMT3B, and EZH2. [Agilent-014707]: Two-condition: untreated SW480 cells (U) vs H2O2 treated SW480 cells (T). Five-mark: SIRT1, gamma-HA2X, DNMT1, DNMT3B, and EZH2. [Agilent-026595]: Two-condition: untreated SW480 cells (U) vs H2O2 treated SW480 cells (T). Five-mark: SIRT1, gamma-HA2X, DNMT1, DNMT3B, and H3. [Agilent-027811]: Two-condition: untreated SW480 cells (U) vs H2O2 treated SW480 cells (T). Four-mark: H3, 3MeK4H3, AcK16H4, and 3MeK27H3.
2011-12-01 | E-GEOD-32382 | biostudies-arrayexpress
Project description:Exosomes are released by most cells to the extracellular environment, and are involved in cell-to-cell-communication. Exosomes contain specific repertoires of mRNAs, miRNAs and other non-coding RNAs that can be functionally transferred to recipient cells. However, the mechanisms that control the specific loading of RNA species into exosomes remain unknown. Here we describe short sequence motifs present in miRNAs that control their localization into exosomes. The protein hnRNPA2B1 specifically binds exosomal miRNAs through the recognition of these motifs and controls their loading into exosomes. Moreover, hnRNPA2B1 in exosomes is sumoylated, and sumoylation controls the binding of hnRNPA2B1 to miRNAs. The loading of miRNAs into exosomes can be modulated by mutagenesis of the identified motifs or changes in hnRNPA2B1 expression levels. These findings identify hnRNPA2B1 as a key player in miRNA sorting into exosomes and provide potential tools for the packaging of selected regulatory RNAs into exosomes and their use in biomedical applications.
2014-04-16 | PXD000350 | Pride
Project description:Extracellular vesicles (EVs) are mammalian cell-derived nano-scale structures enclosed by a lipid bilayer that were previously considered to be cell debris with little biological value. However, EVs are now recognized to possess biological function, acting as a packaging, transport and delivery mechanism by which functional molecules (i.e. miRNAs) can be transferred to target cells over some distance. To examine the miRNA from keratinocyte-derived EVs, we isolated three distinct populations of EVs from both HaCaT and primary human keratinocytes (PKCs) and characterized their biophysical, biochemical and functional features by using microscopy, immunoblotting, nanoparticle tracking, and next generation sequencing. We identified 1048; 906; and, 704 miRNAs, respectively, in apoptotic bodies (APs), microvesicles (MVs) and exosomes (EXs) released from HaCaT, and 608; 506; and, 622 miRNAs in APs, MVs and EXs released from PKCs. In which, there were 623 and 437 identified miRNAs common to three HaCaT-derived EVs and PKC-derived EVs, respectively. In addition, we found hundreds of exosomal miRNAs that were previously un-reported and that differences in the abundance levels of the identified EV miRNAs could discriminate between the three EV populations. These data contribute to EV-identified miRNA database, especially keratinocyte-derived EV miRNA content.
2019-05-20 | GSE106453 | GEO
Project description:ChIP-on-chip tilling array comparing untreated human SW480 cells vs SW480 cells treated with 2mM H2O2 for 30min. Exposing cells to the reactive oxygen species, hydrogen peroxide, recruits DNA methyltransferase 1 (DNMT1) to damaged chromatin. DNMT1 becomes part of a complex(es) containing DNMT3B and members of Polycomb Repressive Complex 4. The goal was to determine the effect of hydrogen peroxide treatment on chromatin, including changes in histone modifications and binding patterns of chromatin-associated proteins.
2011-12-01 | GSE32382 | GEO
Project description:DNA methylation is an important regulatory mechanism that contributes to transcriptional repression. To examine whether DNA methylation affects the transcriptional changes of DNA repair genes under H2O2-induced oxidative stress, we performed reduced representation bisulfite sequencing (RRBS) in H2O2-treated and untreated HCT116 cells.
2016-05-01 | GSE75963 | GEO
Project description:Foxp3+ regulatory T (Treg) cells prevent inflammatory disease but the mechanistic basis of suppression is not understood completely . Gene silencing by RNA interference can act in a cell-autonomous and non-cell-autonomous manner, providing mechanisms of inter-cellular regulation. Here, we demonstrate that non-cell-autonomous gene silencing, mediated by miRNA-containing exosomes, is a mechanism employed by Treg cells to suppress T cell-mediated disease. Treg cells transferred microRNAs (miRNA) to various immune cells, including T helper 1 (Th1) cells, suppressing Th1 cell proliferation and cytokine secretion. Use of Dicer-deficient or Rab27a and Rab27b double-deficient Treg cells to disrupt miRNA-biogenesis or the exosomal pathway, respectively, established a requirement for miRNAs and exosomes for Treg cell-mediated suppression. Transcriptional analysis and miRNA inhibitor studies showed that exosome-mediated transfer of Let-7d from Treg cell to Th1 cells contributed to suppression and prevention of systemic disease. These studies reveal a mechanism of Treg cell-mediated suppression mediated by miRNA-containing exosomes. Regulatory T cells (CD4+CD25hiFoxp3rfp+, Treg) were isolated from naive mice. RNA as extracted form some Treg cells, while others were cultured in complete IMDM media for 3 days, stimulated with anti-CD3 anti-CD3 (1ug/ml) and anti-CD28 (10ug/ml). Exosomes were recovered from Treg cell supernatant, as described, and RNA was extracted form the purified exosomes. To identify which miRNAs were transferred to Dicer-deficient (KO) cells from Treg cells, we cultured Dicer KO cells alone, or co-cultured Dicer KO cells with Treg cells. RNA was extracted form Dicer KO cells cultured alone or from Dicer KO cells cultured in the presence of Treg cells. 3 x biological replicates were used. Each biological replicate was derived from a pool of 3-5 samples.
2014-08-27 | E-GEOD-60615 | biostudies-arrayexpress
Project description:Dendritic cells (DCs) are the most potent antigen (Ag)-presenting cells. Whereas immature DCs down-regulate T cell responses to induce/maintain immunological tolerance, mature DCs promote immunity. To amplify their functions, DCs communicate with neighboring DCs through soluble mediators, cell-to-cell contact and vesicle exchange. Transfer of nanovesicles (<100nm) derived from the endocytic pathway (termed exosomes) represents a novel mechanism of DC-to-DC communication. The facts that exosomes contain exosome-shuttle microRNAs (miRNAs), and DC functions can be regulated by exogenous miRNAs, suggest that DC-to-DC interactions could be mediated through exosome-shuttle miRNAs, an hypothesis that remains to be tested. Importantly, the mechanism of transfer of exosome-shuttle miRNAs from the exosome lumen to the cytosol of target cells is unknown. Here, we demonstrate that DCs release exosomes with different miRNAs depending on the maturation of the DCs. By visualizing spontaneous transfer of exosomes between DCs, we demonstrate that exosomes fused with the target DCs, the latter followed by release of the exosome content into the DC cytosol. Importantly, exosome-shuttle miRNAs are functional, as they repress target mRNAs of acceptor DCs. Our findings unveil a mechanism of transfer of exosome-shuttle miRNAs between DCs and its role as a means of communication and post-transcriptional regulation between DCs. The study has analyzed the microRNA content of 4 samples of immature exosomes, 4 samples of matures exosomes, 2 samples of immature bone-marrow-derived DCs, and 2 samples of mature bone marrow-derived DCs.
2011-10-24 | E-GEOD-33179 | biostudies-arrayexpress
Project description:More and more studies have showed that plasma exosomal miRNAs are biomarkers for disease. The aim of the study were to investigate the miRNA profiling in plasma exosomes of patients with segmental vitiligo (SV) and to find biomarkers in plasma exosomes for patients with SV. Plasma exosomes and exosomal RNA of 7 SV patients and 8 health persons were purified by exoRNeasy Serum/Plasma Maxi Kit. The miRNA profiles of the 15 samples were sequenced using HiSeq 2500 (Illumina) and analyzed by Reads Per Million (RPM) values and edgeR algorithm. Some differently expressed miRNAs in plasma exosomes and skin tissues of the two sets were validated by qRT–PCR.A total of 85 miRNAs in plasma exosomes showed differential expression between SV patients and health persons, with a |log2（Fold Change）|≥1 and P-value < 0.05. Several miRNAs were confirmed by qRT–PCR and showed similar expression patterns between plasma exosomes and skin tissues. Our study depict the miRNAs expression profiles in plasma exosomes of SV patients and suggest that several miRNAs in plasma exosomes may serve as biomarkers for SV.
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