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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 VEGF and miR-200b expression in HRMECs.A: HRMECs exposed to various concentrations of D-glucose for 24 hours exhibited differential mRNA levels of VEGF. Compared to 5mM D-glucose, VEGF expression was significantly increased at 15mM and 25mM D-glucose concentrations, with no change at 20mM L-glucose. B: Measured by WST-1 assay, HRMECs exposed to increasing concentrations of D-glucose for 24 hours exhibited decreased cell viability at 25mM, 50mM and 100mM compared to 5mM. C: HRMECs exposed to 25mM (high glucose; HG) glucose for 24 and 48 hours demonstrated significantly increased VEGF mRNA compared to 5mM (normal glucose; NG). These differences were not observed at time points earlier than 24 hours. D,E: HRMECs exposed to 5mM D-glucose (NG) 25mM D-glucose (HG) and 20mM L-glucose+5mM D-glucose (osmotic control; OSM). HRMECs cultured for 24 hours and 48 hours in HG showed significantly decreased levels of miR-200b with parallel increased levels of VEGF expression compared to NG and OSM. F,G: VEGF is also increased at the protein level in HG compared to NG as measured by Western Blotting. [* p < 0.05 compared to NG; n = 6; data expressed as mean ± SEM, normalized to β-actin or U6 and expressed as a fold change of NG].
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pone.0123987.g001: High levels of glucose alter VEGF and miR-200b expression in HRMECs.A: HRMECs exposed to various concentrations of D-glucose for 24 hours exhibited differential mRNA levels of VEGF. Compared to 5mM D-glucose, VEGF expression was significantly increased at 15mM and 25mM D-glucose concentrations, with no change at 20mM L-glucose. B: Measured by WST-1 assay, HRMECs exposed to increasing concentrations of D-glucose for 24 hours exhibited decreased cell viability at 25mM, 50mM and 100mM compared to 5mM. C: HRMECs exposed to 25mM (high glucose; HG) glucose for 24 and 48 hours demonstrated significantly increased VEGF mRNA compared to 5mM (normal glucose; NG). These differences were not observed at time points earlier than 24 hours. D,E: HRMECs exposed to 5mM D-glucose (NG) 25mM D-glucose (HG) and 20mM L-glucose+5mM D-glucose (osmotic control; OSM). HRMECs cultured for 24 hours and 48 hours in HG showed significantly decreased levels of miR-200b with parallel increased levels of VEGF expression compared to NG and OSM. F,G: VEGF is also increased at the protein level in HG compared to NG as measured by Western Blotting. [* p < 0.05 compared to NG; n = 6; data expressed as mean ± SEM, normalized to β-actin or U6 and expressed as a fold change of NG].

Mentions: This was one of the first investigations to expose the human retinal microvascular endothelial cell (HRMEC) type to high glucose. Therefore, initial experiments were performed to confirm whether glucose increases VEGF levels. L-glucose treatment (osmotic control; OSM) was also used. L-glucose is metabolically inactive and does not cause oxidative stress and hyperglycemia-induced signaling changes as D-glucose does [15,16,40]. We show that VEGF mRNA levels were significantly increased after 24 hour exposure to medium containing 25mM glucose compared to 5mM glucose, while concentrations greater than 25mM did not show any differences in VEGF expression levels (Fig 1A). 20mM L-glucose did not have any effect on VEGF expression (Fig 1A). Furthermore, increasing glucose cocnentrations showed increased cytotoxicity as measured by a WST-1 assay (Fig 1B). Therefore, 5mM glucose was selected as normal glucose (NG) and 25mM glucose was selected as the high glucose (HG) for all subsequent analyses. To test the effects of glucose at different time points, HRMECs were exposed to 5mM or 25mM D-glucose for various durations. VEGF transcript levels were significantly increased in HG compared to NG at 24 hour and 48 hours time points, though no significant changes were observed before these time points (Fig 1C). Therefore, subsequent experiments were performed at these time points to test the effects of glucose exposure on endothelial cells. These findings are consistent with previous studies performed in our laboratory using various endothelial cell types.


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 VEGF and miR-200b expression in HRMECs.A: HRMECs exposed to various concentrations of D-glucose for 24 hours exhibited differential mRNA levels of VEGF. Compared to 5mM D-glucose, VEGF expression was significantly increased at 15mM and 25mM D-glucose concentrations, with no change at 20mM L-glucose. B: Measured by WST-1 assay, HRMECs exposed to increasing concentrations of D-glucose for 24 hours exhibited decreased cell viability at 25mM, 50mM and 100mM compared to 5mM. C: HRMECs exposed to 25mM (high glucose; HG) glucose for 24 and 48 hours demonstrated significantly increased VEGF mRNA compared to 5mM (normal glucose; NG). These differences were not observed at time points earlier than 24 hours. D,E: HRMECs exposed to 5mM D-glucose (NG) 25mM D-glucose (HG) and 20mM L-glucose+5mM D-glucose (osmotic control; OSM). HRMECs cultured for 24 hours and 48 hours in HG showed significantly decreased levels of miR-200b with parallel increased levels of VEGF expression compared to NG and OSM. F,G: VEGF is also increased at the protein level in HG compared to NG as measured by Western Blotting. [* p < 0.05 compared to NG; n = 6; data expressed as mean ± SEM, normalized to β-actin or U6 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.g001: High levels of glucose alter VEGF and miR-200b expression in HRMECs.A: HRMECs exposed to various concentrations of D-glucose for 24 hours exhibited differential mRNA levels of VEGF. Compared to 5mM D-glucose, VEGF expression was significantly increased at 15mM and 25mM D-glucose concentrations, with no change at 20mM L-glucose. B: Measured by WST-1 assay, HRMECs exposed to increasing concentrations of D-glucose for 24 hours exhibited decreased cell viability at 25mM, 50mM and 100mM compared to 5mM. C: HRMECs exposed to 25mM (high glucose; HG) glucose for 24 and 48 hours demonstrated significantly increased VEGF mRNA compared to 5mM (normal glucose; NG). These differences were not observed at time points earlier than 24 hours. D,E: HRMECs exposed to 5mM D-glucose (NG) 25mM D-glucose (HG) and 20mM L-glucose+5mM D-glucose (osmotic control; OSM). HRMECs cultured for 24 hours and 48 hours in HG showed significantly decreased levels of miR-200b with parallel increased levels of VEGF expression compared to NG and OSM. F,G: VEGF is also increased at the protein level in HG compared to NG as measured by Western Blotting. [* p < 0.05 compared to NG; n = 6; data expressed as mean ± SEM, normalized to β-actin or U6 and expressed as a fold change of NG].
Mentions: This was one of the first investigations to expose the human retinal microvascular endothelial cell (HRMEC) type to high glucose. Therefore, initial experiments were performed to confirm whether glucose increases VEGF levels. L-glucose treatment (osmotic control; OSM) was also used. L-glucose is metabolically inactive and does not cause oxidative stress and hyperglycemia-induced signaling changes as D-glucose does [15,16,40]. We show that VEGF mRNA levels were significantly increased after 24 hour exposure to medium containing 25mM glucose compared to 5mM glucose, while concentrations greater than 25mM did not show any differences in VEGF expression levels (Fig 1A). 20mM L-glucose did not have any effect on VEGF expression (Fig 1A). Furthermore, increasing glucose cocnentrations showed increased cytotoxicity as measured by a WST-1 assay (Fig 1B). Therefore, 5mM glucose was selected as normal glucose (NG) and 25mM glucose was selected as the high glucose (HG) for all subsequent analyses. To test the effects of glucose at different time points, HRMECs were exposed to 5mM or 25mM D-glucose for various durations. VEGF transcript levels were significantly increased in HG compared to NG at 24 hour and 48 hours time points, though no significant changes were observed before these time points (Fig 1C). Therefore, subsequent experiments were performed at these time points to test the effects of glucose exposure on endothelial cells. These findings are consistent with previous studies performed in our laboratory using various endothelial cell types.

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