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Polycomb repressive complex 2 regulates MiR-200b in retinal endothelial cells: potential relevance in diabetic retinopathy.

Ruiz MA, Feng B, Chakrabarti S - PLoS ONE (2015)

Bottom Line: Histone methyltransferase complex, Polycomb Repressive Complex 2 (PRC2), has been shown to repress miRNAs in neoplastic process.We show that human retinal microvascular endothelial cells exposed to high levels of glucose regulate miR-200b repression through histone methylation and that inhibition of PRC2 increases miR-200b while reducing VEGF.This research established a repressive relationship between PRC2 and miR-200b, providing evidence of a novel mechanism of miRNA regulation through histone methylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, Western University, London, Ontario, Canada.

ABSTRACT
Glucose-induced augmented vascular endothelial growth factor (VEGF) production is a key event in diabetic retinopathy. We have previously demonstrated that downregulation of miR-200b increases VEGF, mediating structural and functional changes in the retina in diabetes. However, mechanisms regulating miR-200b in diabetes are not known. Histone methyltransferase complex, Polycomb Repressive Complex 2 (PRC2), has been shown to repress miRNAs in neoplastic process. We hypothesized that, in diabetes, PRC2 represses miR-200b through its histone H3 lysine-27 trimethylation mark. We show that human retinal microvascular endothelial cells exposed to high levels of glucose regulate miR-200b repression through histone methylation and that inhibition of PRC2 increases miR-200b while reducing VEGF. Furthermore, retinal tissue from animal models of diabetes showed increased expression of major PRC2 components, demonstrating in vivo relevance. This research established a repressive relationship between PRC2 and miR-200b, providing evidence of a novel mechanism of miRNA regulation through histone methylation.

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High levels of glucose alter H3K27me3 and RNA polymerase 2 association at the miR-200b promoter region.A: ChIP-qPCR analysis of H3K27 trimethylation (H3K27me3) at the miR-200b promoter region. Association of H3K27me3 at the miR-200b promoter region was significantly increased in 25mM (HG) glucose compared to 5mM (NG) glucose. B: ChIP-qPCR analysis of RNA polymerase 2 (Pol2) at the miR-200b promoter region. Association of Pol2 at the miR-200b promoter region was significantly decreased in HG compared to 5mM NG. C: ChIP-qPCR analysis of H3K27me3 at the human alpha-satelite region. Association of H3K27me3 at the human alpha satellite region showed no significant change between HG and NG. D: ChIP-qPCR analysis of Pol2 at the GAPDH promoter region. Association of Pol2 at the GAPDH promoter region showed no significant difference between 25mM (HG) glucose and 5mM (NG) glucose. Association of IgG and ascitic fluid (negative control) was minimal and showed no difference between NG and HG in all experiments. [* p < 0.05 compared to NG; n = 3; data expressed as mean percentage of input ± SEM].
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pone.0123987.g003: High levels of glucose alter H3K27me3 and RNA polymerase 2 association at the miR-200b promoter region.A: ChIP-qPCR analysis of H3K27 trimethylation (H3K27me3) at the miR-200b promoter region. Association of H3K27me3 at the miR-200b promoter region was significantly increased in 25mM (HG) glucose compared to 5mM (NG) glucose. B: ChIP-qPCR analysis of RNA polymerase 2 (Pol2) at the miR-200b promoter region. Association of Pol2 at the miR-200b promoter region was significantly decreased in HG compared to 5mM NG. C: ChIP-qPCR analysis of H3K27me3 at the human alpha-satelite region. Association of H3K27me3 at the human alpha satellite region showed no significant change between HG and NG. D: ChIP-qPCR analysis of Pol2 at the GAPDH promoter region. Association of Pol2 at the GAPDH promoter region showed no significant difference between 25mM (HG) glucose and 5mM (NG) glucose. Association of IgG and ascitic fluid (negative control) was minimal and showed no difference between NG and HG in all experiments. [* p < 0.05 compared to NG; n = 3; data expressed as mean percentage of input ± SEM].

Mentions: Following ChIP-qPCR analysis, H3K27me3 was found to be significantly increased at the miR-200b promoter region in HG compared to NG controls (Fig 3A). IgG isotype control pull down showed significantly less association than the H3K27me3-specific antibody. Also, Pol2 was found to be significantly decreased at the miR-200b promoter region in HG compared to NG controls (Fig 3B). Again, specificity of this antibody was demonstrated as association with the negative control was minimal. Therefore, this increase of the H3K27me3, which is specific to the PRC2 complex, demonstrates a strong regulatory relationship between PRC2 and miR-200b at the level of chromatin modification. Furthermore, since this type of methylation is believed to be repressing and is associated with closed chromatin, the observation of decreased Pol2 association at the miR-200b promoter region further supports the mechanism and accounts for the decrease in miR-200b expression demonstrated earlier. Altogether, these results paint a picture of the genomic events occurring at the miR-200b promoter region in HG, and further support that PRC2 may regulate miR-200b in diabetes.


Polycomb repressive complex 2 regulates MiR-200b in retinal endothelial cells: potential relevance in diabetic retinopathy.

Ruiz MA, Feng B, Chakrabarti S - PLoS ONE (2015)

High levels of glucose alter H3K27me3 and RNA polymerase 2 association at the miR-200b promoter region.A: ChIP-qPCR analysis of H3K27 trimethylation (H3K27me3) at the miR-200b promoter region. Association of H3K27me3 at the miR-200b promoter region was significantly increased in 25mM (HG) glucose compared to 5mM (NG) glucose. B: ChIP-qPCR analysis of RNA polymerase 2 (Pol2) at the miR-200b promoter region. Association of Pol2 at the miR-200b promoter region was significantly decreased in HG compared to 5mM NG. C: ChIP-qPCR analysis of H3K27me3 at the human alpha-satelite region. Association of H3K27me3 at the human alpha satellite region showed no significant change between HG and NG. D: ChIP-qPCR analysis of Pol2 at the GAPDH promoter region. Association of Pol2 at the GAPDH promoter region showed no significant difference between 25mM (HG) glucose and 5mM (NG) glucose. Association of IgG and ascitic fluid (negative control) was minimal and showed no difference between NG and HG in all experiments. [* p < 0.05 compared to NG; n = 3; data expressed as mean percentage of input ± SEM].
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4401764&req=5

pone.0123987.g003: High levels of glucose alter H3K27me3 and RNA polymerase 2 association at the miR-200b promoter region.A: ChIP-qPCR analysis of H3K27 trimethylation (H3K27me3) at the miR-200b promoter region. Association of H3K27me3 at the miR-200b promoter region was significantly increased in 25mM (HG) glucose compared to 5mM (NG) glucose. B: ChIP-qPCR analysis of RNA polymerase 2 (Pol2) at the miR-200b promoter region. Association of Pol2 at the miR-200b promoter region was significantly decreased in HG compared to 5mM NG. C: ChIP-qPCR analysis of H3K27me3 at the human alpha-satelite region. Association of H3K27me3 at the human alpha satellite region showed no significant change between HG and NG. D: ChIP-qPCR analysis of Pol2 at the GAPDH promoter region. Association of Pol2 at the GAPDH promoter region showed no significant difference between 25mM (HG) glucose and 5mM (NG) glucose. Association of IgG and ascitic fluid (negative control) was minimal and showed no difference between NG and HG in all experiments. [* p < 0.05 compared to NG; n = 3; data expressed as mean percentage of input ± SEM].
Mentions: Following ChIP-qPCR analysis, H3K27me3 was found to be significantly increased at the miR-200b promoter region in HG compared to NG controls (Fig 3A). IgG isotype control pull down showed significantly less association than the H3K27me3-specific antibody. Also, Pol2 was found to be significantly decreased at the miR-200b promoter region in HG compared to NG controls (Fig 3B). Again, specificity of this antibody was demonstrated as association with the negative control was minimal. Therefore, this increase of the H3K27me3, which is specific to the PRC2 complex, demonstrates a strong regulatory relationship between PRC2 and miR-200b at the level of chromatin modification. Furthermore, since this type of methylation is believed to be repressing and is associated with closed chromatin, the observation of decreased Pol2 association at the miR-200b promoter region further supports the mechanism and accounts for the decrease in miR-200b expression demonstrated earlier. Altogether, these results paint a picture of the genomic events occurring at the miR-200b promoter region in HG, and further support that PRC2 may regulate miR-200b in diabetes.

Bottom Line: Histone methyltransferase complex, Polycomb Repressive Complex 2 (PRC2), has been shown to repress miRNAs in neoplastic process.We show that human retinal microvascular endothelial cells exposed to high levels of glucose regulate miR-200b repression through histone methylation and that inhibition of PRC2 increases miR-200b while reducing VEGF.This research established a repressive relationship between PRC2 and miR-200b, providing evidence of a novel mechanism of miRNA regulation through histone methylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, Western University, London, Ontario, Canada.

ABSTRACT
Glucose-induced augmented vascular endothelial growth factor (VEGF) production is a key event in diabetic retinopathy. We have previously demonstrated that downregulation of miR-200b increases VEGF, mediating structural and functional changes in the retina in diabetes. However, mechanisms regulating miR-200b in diabetes are not known. Histone methyltransferase complex, Polycomb Repressive Complex 2 (PRC2), has been shown to repress miRNAs in neoplastic process. We hypothesized that, in diabetes, PRC2 represses miR-200b through its histone H3 lysine-27 trimethylation mark. We show that human retinal microvascular endothelial cells exposed to high levels of glucose regulate miR-200b repression through histone methylation and that inhibition of PRC2 increases miR-200b while reducing VEGF. Furthermore, retinal tissue from animal models of diabetes showed increased expression of major PRC2 components, demonstrating in vivo relevance. This research established a repressive relationship between PRC2 and miR-200b, providing evidence of a novel mechanism of miRNA regulation through histone methylation.

Show MeSH
Related in: MedlinePlus