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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 SUZ12 siRNA demonstrate increased miR-200b expression, decreased VEGF expression and decreased endothelial branching.A,B: Gene knockdown of EZH2 and SUZ12 was confirmed by qPCR. C,D: In HRMECs transfected with EZH2 siRNA in HG, miR-200b and VEGF were not significantly different from HG+control siRNA but decreased compared to NG+control siRNA. In HRMECs transfected with SUZ12 siRNA in HG, miR-200b was significantly increased with decreased levels of VEGF compared to HRMECs transfected with control siRNA, with levels similar to NG+control siRNA. E,F: Tube formation assay to measure endothelial branching. HRMECs transfected with HG+control siRNA demonstrated significantly increased branching compared to NG+control siRNA. Transfection of EZH2 siRNA did not reduce endothelial branching significantly compared to HG+control siRNA. However, transfection of SUZ12 siRNA significantly reduced endothelial branching compared to HG+control siRNA. [NG+control siRNA = 5mM D-glucose + 100nM control siRNA, HG+control siRNA = 25mM D-glucose + 100nM control siRNA, HG+EZH2 siRNA = 25mM D-glucose + 100nM EZH2 siRNA, HG+SUZ12 siRNA = 25mM + 100nM SUZ12 siRNA; identical letters represent groups that are not significantly different; p < 0.05; n = 6; data expressed as mean ± SEM, normalized to U6 or β-actin and expressed as a fold change of NG+control siRNA].
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pone.0123987.g005: HRMECs treated with SUZ12 siRNA demonstrate increased miR-200b expression, decreased VEGF expression and decreased endothelial branching.A,B: Gene knockdown of EZH2 and SUZ12 was confirmed by qPCR. C,D: In HRMECs transfected with EZH2 siRNA in HG, miR-200b and VEGF were not significantly different from HG+control siRNA but decreased compared to NG+control siRNA. In HRMECs transfected with SUZ12 siRNA in HG, miR-200b was significantly increased with decreased levels of VEGF compared to HRMECs transfected with control siRNA, with levels similar to NG+control siRNA. E,F: Tube formation assay to measure endothelial branching. HRMECs transfected with HG+control siRNA demonstrated significantly increased branching compared to NG+control siRNA. Transfection of EZH2 siRNA did not reduce endothelial branching significantly compared to HG+control siRNA. However, transfection of SUZ12 siRNA significantly reduced endothelial branching compared to HG+control siRNA. [NG+control siRNA = 5mM D-glucose + 100nM control siRNA, HG+control siRNA = 25mM D-glucose + 100nM control siRNA, HG+EZH2 siRNA = 25mM D-glucose + 100nM EZH2 siRNA, HG+SUZ12 siRNA = 25mM + 100nM SUZ12 siRNA; identical letters represent groups that are not significantly different; p < 0.05; n = 6; data expressed as mean ± SEM, normalized to U6 or β-actin and expressed as a fold change of NG+control siRNA].

Mentions: To further eludicate the role of specific components of PRC2 in the regulation of miR-200b, siRNA-mediated gene knockdown of EZH2 and SUZ12 was performed. Knockdown efficiency was verified by RT-PCR (Fig 5A and 5B). In HG, HRMECs transfected with control siRNA showed a significant decrease in miR-200b expression and increase in VEGF expression (Fig 5C and 5D). When EZH2 was silenced in HG, miR-200b and VEGF showed no differences in expression compared to HRMECs treated with control siRNA in HG (Fig 5C and 5D). However, silencing of SUZ12 increased miR-200b and decreased VEGF, with levels similar to HRMECs transfected with control siRNA in NG (Fig 5C and 5D). In addition, a tube formation assay was conducted to provide a functional correlate. HRMECs transfected with control siRNA and EZH2 siRNA in HG showed significantly increased branching compared to HRMECs transfected with control siRNA in NG (Fig 5E and 5F). HRMECs treated with SUZ12 siRNA in HG showed decreased branching, equivalent to HRMECs treated with control siRNA in NG (Fig 5E and 5F). Altogether, this data suggests that SUZ12 is important in regulating miR-200b in HG, as knockdown of SUZ12 corrected miR-200b and VEGF levels.


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 SUZ12 siRNA demonstrate increased miR-200b expression, decreased VEGF expression and decreased endothelial branching.A,B: Gene knockdown of EZH2 and SUZ12 was confirmed by qPCR. C,D: In HRMECs transfected with EZH2 siRNA in HG, miR-200b and VEGF were not significantly different from HG+control siRNA but decreased compared to NG+control siRNA. In HRMECs transfected with SUZ12 siRNA in HG, miR-200b was significantly increased with decreased levels of VEGF compared to HRMECs transfected with control siRNA, with levels similar to NG+control siRNA. E,F: Tube formation assay to measure endothelial branching. HRMECs transfected with HG+control siRNA demonstrated significantly increased branching compared to NG+control siRNA. Transfection of EZH2 siRNA did not reduce endothelial branching significantly compared to HG+control siRNA. However, transfection of SUZ12 siRNA significantly reduced endothelial branching compared to HG+control siRNA. [NG+control siRNA = 5mM D-glucose + 100nM control siRNA, HG+control siRNA = 25mM D-glucose + 100nM control siRNA, HG+EZH2 siRNA = 25mM D-glucose + 100nM EZH2 siRNA, HG+SUZ12 siRNA = 25mM + 100nM SUZ12 siRNA; identical letters represent groups that are not significantly different; p < 0.05; n = 6; data expressed as mean ± SEM, normalized to U6 or β-actin and expressed as a fold change of NG+control siRNA].
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pone.0123987.g005: HRMECs treated with SUZ12 siRNA demonstrate increased miR-200b expression, decreased VEGF expression and decreased endothelial branching.A,B: Gene knockdown of EZH2 and SUZ12 was confirmed by qPCR. C,D: In HRMECs transfected with EZH2 siRNA in HG, miR-200b and VEGF were not significantly different from HG+control siRNA but decreased compared to NG+control siRNA. In HRMECs transfected with SUZ12 siRNA in HG, miR-200b was significantly increased with decreased levels of VEGF compared to HRMECs transfected with control siRNA, with levels similar to NG+control siRNA. E,F: Tube formation assay to measure endothelial branching. HRMECs transfected with HG+control siRNA demonstrated significantly increased branching compared to NG+control siRNA. Transfection of EZH2 siRNA did not reduce endothelial branching significantly compared to HG+control siRNA. However, transfection of SUZ12 siRNA significantly reduced endothelial branching compared to HG+control siRNA. [NG+control siRNA = 5mM D-glucose + 100nM control siRNA, HG+control siRNA = 25mM D-glucose + 100nM control siRNA, HG+EZH2 siRNA = 25mM D-glucose + 100nM EZH2 siRNA, HG+SUZ12 siRNA = 25mM + 100nM SUZ12 siRNA; identical letters represent groups that are not significantly different; p < 0.05; n = 6; data expressed as mean ± SEM, normalized to U6 or β-actin and expressed as a fold change of NG+control siRNA].
Mentions: To further eludicate the role of specific components of PRC2 in the regulation of miR-200b, siRNA-mediated gene knockdown of EZH2 and SUZ12 was performed. Knockdown efficiency was verified by RT-PCR (Fig 5A and 5B). In HG, HRMECs transfected with control siRNA showed a significant decrease in miR-200b expression and increase in VEGF expression (Fig 5C and 5D). When EZH2 was silenced in HG, miR-200b and VEGF showed no differences in expression compared to HRMECs treated with control siRNA in HG (Fig 5C and 5D). However, silencing of SUZ12 increased miR-200b and decreased VEGF, with levels similar to HRMECs transfected with control siRNA in NG (Fig 5C and 5D). In addition, a tube formation assay was conducted to provide a functional correlate. HRMECs transfected with control siRNA and EZH2 siRNA in HG showed significantly increased branching compared to HRMECs transfected with control siRNA in NG (Fig 5E and 5F). HRMECs treated with SUZ12 siRNA in HG showed decreased branching, equivalent to HRMECs treated with control siRNA in NG (Fig 5E and 5F). Altogether, this data suggests that SUZ12 is important in regulating miR-200b in HG, as knockdown of SUZ12 corrected miR-200b and VEGF levels.

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