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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 the expressions of PRC2 component and related genes.Real time RT-PCR analysis of PRC2 components in HRMECs exposed to 5mM D-glucose (NG), 25mM D-glucose (HG), and 20mM L-glucose+5mM D-glucose (OSM). A: After 24 and 48 hour exposure to D-glucose, levels of EZH2, EED and SUZ12, were significantly increased compared to NG and OSM controls. B: After 24 exposure to D-glucose, KDM6A and KDM6B levels did not change in HG. After 48 hours, KDM6A was significantly decreased compared to NG and OSM controls, while KDM6B showed no change. [* p < 0.05 compared to NG; n = 6; data expressed as mean ± SEM, normalized to β-actin and expressed as a fold change of NG].
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pone.0123987.g002: High levels of glucose alter the expressions of PRC2 component and related genes.Real time RT-PCR analysis of PRC2 components in HRMECs exposed to 5mM D-glucose (NG), 25mM D-glucose (HG), and 20mM L-glucose+5mM D-glucose (OSM). A: After 24 and 48 hour exposure to D-glucose, levels of EZH2, EED and SUZ12, were significantly increased compared to NG and OSM controls. B: After 24 exposure to D-glucose, KDM6A and KDM6B levels did not change in HG. After 48 hours, KDM6A was significantly decreased compared to NG and OSM controls, while KDM6B showed no change. [* p < 0.05 compared to NG; n = 6; data expressed as mean ± SEM, normalized to β-actin and expressed as a fold change of NG].

Mentions: At both 24 and 48 hours in HG, mRNA of EZH2, EED and SUZ12 were elevated compared to NG and OSM controls (Fig 2A, 2B and 2C). These observations show increased expression of the PRC2 components, which were hypothesized to negatively regulate miR-200b expression. Furthermore, no difference was observed in expression levels of KDM6A and KDM6B between NG, HG and OSM controls (Fig 2D and 2E). Interestingly, KDM6A was observed to significantly decrease in HG at the 48 hour time point (Fig 2D). This suggests there is no change or even a decrease in demethylase expression for H3K27 methylation. Altogether, an increase in expression of PRC2 with no change or decrease in demethylase expression suggests a shift towards increase of H3K27me3 in HG. Overall, these experiments provide correlational evidence linking PRC2 to negatively regulating miR-200b in HG, though additional supporting evidence is necessary.


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 the expressions of PRC2 component and related genes.Real time RT-PCR analysis of PRC2 components in HRMECs exposed to 5mM D-glucose (NG), 25mM D-glucose (HG), and 20mM L-glucose+5mM D-glucose (OSM). A: After 24 and 48 hour exposure to D-glucose, levels of EZH2, EED and SUZ12, were significantly increased compared to NG and OSM controls. B: After 24 exposure to D-glucose, KDM6A and KDM6B levels did not change in HG. After 48 hours, KDM6A was significantly decreased compared to NG and OSM controls, while KDM6B showed no change. [* p < 0.05 compared to NG; n = 6; data expressed as mean ± SEM, normalized to β-actin and expressed as a fold change of NG].
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4401764&req=5

pone.0123987.g002: High levels of glucose alter the expressions of PRC2 component and related genes.Real time RT-PCR analysis of PRC2 components in HRMECs exposed to 5mM D-glucose (NG), 25mM D-glucose (HG), and 20mM L-glucose+5mM D-glucose (OSM). A: After 24 and 48 hour exposure to D-glucose, levels of EZH2, EED and SUZ12, were significantly increased compared to NG and OSM controls. B: After 24 exposure to D-glucose, KDM6A and KDM6B levels did not change in HG. After 48 hours, KDM6A was significantly decreased compared to NG and OSM controls, while KDM6B showed no change. [* p < 0.05 compared to NG; n = 6; data expressed as mean ± SEM, normalized to β-actin and expressed as a fold change of NG].
Mentions: At both 24 and 48 hours in HG, mRNA of EZH2, EED and SUZ12 were elevated compared to NG and OSM controls (Fig 2A, 2B and 2C). These observations show increased expression of the PRC2 components, which were hypothesized to negatively regulate miR-200b expression. Furthermore, no difference was observed in expression levels of KDM6A and KDM6B between NG, HG and OSM controls (Fig 2D and 2E). Interestingly, KDM6A was observed to significantly decrease in HG at the 48 hour time point (Fig 2D). This suggests there is no change or even a decrease in demethylase expression for H3K27 methylation. Altogether, an increase in expression of PRC2 with no change or decrease in demethylase expression suggests a shift towards increase of H3K27me3 in HG. Overall, these experiments provide correlational evidence linking PRC2 to negatively regulating miR-200b in HG, though additional supporting evidence is necessary.

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