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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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HRMECs treated with 3-Deazaneplanocin A (DZNep) chemical inhibitor demonstrated increased miR-200b and decreased VEGF expression.A: After 24 exposure to DZNep in HG, miR-200b levels were significantly increased. B,C: In parallel levels of VEGF mRNA and secreted VEGF protein were decreased in HRMECs treated in HG with DZNep compared to controls. [NG = 5mM D-glucose, HG = 25mM D-glucose, NG+DMSO = 5mM D-glucose + 0.05% DMSO, HG+DMSO = 25mM D-glucose + 0.05% DMSO, NG+DZNEP = 5mM D-glucose + 5μM DZNep, HG+DZNEP = 25mM + 5μM DZNep; identical letters represent groups that are not significantly different; p < 0.05; n = 6; data expressed as mean ± SEM, normalized to β-actin for VEGF and U6 for miR-200b, expressed as a fold change of NG].
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pone.0123987.g004: HRMECs treated with 3-Deazaneplanocin A (DZNep) chemical inhibitor demonstrated increased miR-200b and decreased VEGF expression.A: After 24 exposure to DZNep in HG, miR-200b levels were significantly increased. B,C: In parallel levels of VEGF mRNA and secreted VEGF protein were decreased in HRMECs treated in HG with DZNep compared to controls. [NG = 5mM D-glucose, HG = 25mM D-glucose, NG+DMSO = 5mM D-glucose + 0.05% DMSO, HG+DMSO = 25mM D-glucose + 0.05% DMSO, NG+DZNEP = 5mM D-glucose + 5μM DZNep, HG+DZNEP = 25mM + 5μM DZNep; identical letters represent groups that are not significantly different; p < 0.05; n = 6; data expressed as mean ± SEM, normalized to β-actin for VEGF and U6 for miR-200b, expressed as a fold change of NG].

Mentions: To demonstrate a cause-and-effect relationship between PRC2 and miR-200b, a chemical inhibitor for H3K27me3, DZNep, was used. HRMECs treated for 24 hours with DZNep showed significantly higher miR-200b levels in both NG and HG compared to the other controls (Fig 4A). In parallel, VEGF mRNA was significantly decreased when HRMECs were treated with DZNep compared to all other controls (Fig 4B). Furthermore, VEGF was measured in the supernatant of treated HRMECs (Fig 4C). VEGF protein was elevated in HRMECs treated in HG and HG with DMSO, compared to respective controls, however this elevation was not seen in HRMECs treated with DZNep, demonstrating parallel changes in VEGF mRNA and protein. Since PRC2 was hypothesized to negatively inhibit miR-200b, through its repressing methylation-mark, increased expression of miR-200b would be expected by inhibiting PRC2. Increased miR-200b expression was observed with decreased VEGF mRNA and protein, a target of miR-200b, providing evidence to support the hypothesis by showing a cause-and-effect relationship by using loss-of-inhibition.


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)

HRMECs treated with 3-Deazaneplanocin A (DZNep) chemical inhibitor demonstrated increased miR-200b and decreased VEGF expression.A: After 24 exposure to DZNep in HG, miR-200b levels were significantly increased. B,C: In parallel levels of VEGF mRNA and secreted VEGF protein were decreased in HRMECs treated in HG with DZNep compared to controls. [NG = 5mM D-glucose, HG = 25mM D-glucose, NG+DMSO = 5mM D-glucose + 0.05% DMSO, HG+DMSO = 25mM D-glucose + 0.05% DMSO, NG+DZNEP = 5mM D-glucose + 5μM DZNep, HG+DZNEP = 25mM + 5μM DZNep; identical letters represent groups that are not significantly different; p < 0.05; n = 6; data expressed as mean ± SEM, normalized to β-actin for VEGF and U6 for miR-200b, expressed as a fold change of NG].
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4401764&req=5

pone.0123987.g004: HRMECs treated with 3-Deazaneplanocin A (DZNep) chemical inhibitor demonstrated increased miR-200b and decreased VEGF expression.A: After 24 exposure to DZNep in HG, miR-200b levels were significantly increased. B,C: In parallel levels of VEGF mRNA and secreted VEGF protein were decreased in HRMECs treated in HG with DZNep compared to controls. [NG = 5mM D-glucose, HG = 25mM D-glucose, NG+DMSO = 5mM D-glucose + 0.05% DMSO, HG+DMSO = 25mM D-glucose + 0.05% DMSO, NG+DZNEP = 5mM D-glucose + 5μM DZNep, HG+DZNEP = 25mM + 5μM DZNep; identical letters represent groups that are not significantly different; p < 0.05; n = 6; data expressed as mean ± SEM, normalized to β-actin for VEGF and U6 for miR-200b, expressed as a fold change of NG].
Mentions: To demonstrate a cause-and-effect relationship between PRC2 and miR-200b, a chemical inhibitor for H3K27me3, DZNep, was used. HRMECs treated for 24 hours with DZNep showed significantly higher miR-200b levels in both NG and HG compared to the other controls (Fig 4A). In parallel, VEGF mRNA was significantly decreased when HRMECs were treated with DZNep compared to all other controls (Fig 4B). Furthermore, VEGF was measured in the supernatant of treated HRMECs (Fig 4C). VEGF protein was elevated in HRMECs treated in HG and HG with DMSO, compared to respective controls, however this elevation was not seen in HRMECs treated with DZNep, demonstrating parallel changes in VEGF mRNA and protein. Since PRC2 was hypothesized to negatively inhibit miR-200b, through its repressing methylation-mark, increased expression of miR-200b would be expected by inhibiting PRC2. Increased miR-200b expression was observed with decreased VEGF mRNA and protein, a target of miR-200b, providing evidence to support the hypothesis by showing a cause-and-effect relationship by using loss-of-inhibition.

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