PRJNA309443: HDAC inhibitor SAHA reverses inflammatory gene expression in diabetic endothelial cells
概要: While histone deacetylase (HDAC) inhibitors are thought to regulate gene expression by post-translational modification of histone as well as non-histone proteins. While histone hyperacetylation has long been considered the paradigmatic mechanism of action, recent genome-wide profiles indicate more complex interactions with the epigenome. In particular, HDAC inhibitors also induce histone deacetylation at the promoters of highly active genes, resulting in gene suppression. This was linked to the loss of histone acetyltransferase (HAT) binding. To illustrate pre-clinical utility of the HDAC inhibitor SAHA as a therapeutic, we show reversal of diabetes-associated EP300 target genes in diabetic HAECs of primary origin. These results were confirmed using SAHA, C646 (EP300/CREBBP inhibitor) or EP300 siRNA. These findings suggest the inhibition of gene expression by SAHA is mediated by EP300 function and provide a rationale for clinical trials of safety and efficacy in patients with diabetes.
项目整体设计: Human aortic endothelial cells from a diabetic and non-diabetic individual were stimulated with DMSO (control), SAHA (2 μM, HDAC inhibitor) or C646 (10 μM, EP300 inhibitor) for 12 hours, or EP300 siRNA or non-target siRNA (control) for 4 hours, followed by 48 hours in fresh media. Study performed in triplicate.
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Human aortic endothelial cells (HAEC) from diabetic (CC-2919) and non-diabetic (CC-2535) individuals were maintained in endothelial cell basal growth media-2 (EBM-2, Lonza) with additional EBM-2 growth factors and supplements and with 10% heat-inactivated fetal bovine serum (Gibco). HAECs used in this study were between 5-9 passages. |
| 处理方案: |
Cells were stimulated with DMSO, SAHA (2 μM) or C646 (10 μM) for 12 hours. EP300 knock-down was generated using ON-TARGETplus Human EP300 (2033) siRNA – SMARTpool (L-003486-00-0005, Dharmacon). Cells were cultured in Opti-MEM® (Life Technologies) media with Lipofectamine® RNAiMAX Reagent (13778-075, Life Technologies) with either non-target (ON-TARGETplus Non-targeting Pool, D-001810-10-05, Dharmacon) or 25 nM EP300 siRNA for four hours. Media was replaced with EBM-2 and RNA was harvested after 48 hours. |
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Total RNA was extracted in TRIzol (Invitrogen) and purified using the Direct-zolTM RNA MiniPrep kit (cat. no: R2052, Zymo Research) according to the manufacturers instructions.RNA was quantified on the MultiNA bioanalyzer (Shimadzu).NEBNext® Poly(A) mRNA Magnetic Isolation Module was used to enrich mRNA from 1 μg of total RNA. |
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Libraries were quantified on the MultiNA Bioanalyzer (Shimadzu) and pooled to equimolar ratios for sequencing. |
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poly(A)+ RNA |
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Forward |
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ILLUMINA |
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Illumina HiSeq 2500 |
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Specific |
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Systems approach to the pharmacological actions of HDAC inhibitors reveals EP300 activities and convergent mechanisms of regulation in diabetes.
Epigenetics . 2017-01-01 [PMID:
28886276]
