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miR-146a-Mediated extracellular matrix protein production in chronic diabetes complications.

Feng B, Chen S, McArthur K, Wu Y, Sen S, Ding Q, Feldman RD, Chakrabarti S - Diabetes (2011)

Bottom Line: Cardiac and renal tissues were analyzed from type 1 and type 2 diabetic animals.Additional experiments showed that p300 regulates miR-146a.Similar changes were seen in the retinas, kidneys, and hearts in type 1 and type 2 diabetic animals.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Schulich School of Medicine and Dentistry and the University of Western Ontario, London, Ontario, Canada.

ABSTRACT

Objective: MicroRNAs (miRNAs), through transcriptional regulation, modulate several cellular processes. In diabetes, increased extracellular matrix protein fibronectin (FN) production is known to occur through histone acetylator p300. Here, we investigated the role of miR-146a, an FN-targeting miRNA, on FN production in diabetes and its relationship with p300.

Research design and methods: miR-146a expressions were measured in endothelial cells from large vessels and retinal microvessels in various glucose levels. FN messenger RNA expression and protein levels with or without miR-146a mimic or antagomir transfection were examined. A luciferase assay was performed to detect miR-146a's binding to FN 3'-untranslated region (UTR). Likewise, retinas from type 1 diabetic rats were studied with or without an intravitreal injection of miR-146a mimic. In situ hybridization was used to localize retinal miR-146a. Cardiac and renal tissues were analyzed from type 1 and type 2 diabetic animals.

Results: A total of 25 mmol/L glucose decreased miR-146a expression and increased FN expression compared with 5 mmol/L glucose in both cell types. miR-146a mimic transfection prevented such change, whereas miR-146a antagomir transfection in the cells in 5 mmol/L glucose caused FN upregulation. A luciferase assay confirmed miR-146a's binding to FN 3'-UTR. miR-146a was localized in the retinal endothelial cells and was decreased in diabetes. Intravitreal miR-146a mimic injection restored retinal miR-146a and decreased FN in diabetes. Additional experiments showed that p300 regulates miR-146a. Similar changes were seen in the retinas, kidneys, and hearts in type 1 and type 2 diabetic animals.

Conclusions: These studies showed a novel, glucose-induced molecular mechanism in which miR-146a participates in the transcriptional circuitry regulating extracellular matrix protein production in diabetes.

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miR-146a levels are regulated with p300. In the endothelial cells, 25 mmol/L (25 mM) glucose-induced p300 mRNA upregulation (A) and increased histone acetylation (B) were prevented by p300 shRNA (p300sh) transfection. In the endothelial cells, p300 shRNA also prevented glucose-induced reduced miR-146a production (C) and FN mRNA (D) and FN protein upregulation (E) in the HUVECs. In parallel, diabetes-induced retinal p300 upregulation (G), miR-146a downregulation (H), and FN upregulation (I) were prevented by intravitreal p300 siRNA (p300si) injection. F: No significant differences were seen with respect to p300 mRNA expression when cells in 25 mmol/L glucose were transfected with miR-146a mimic or scrambled (S) mimic. *Significantly different from 5 mmol/L glucose or control (Co); **significantly different from 25 mmol/L glucose or diabetes (Di). miRNA levels are expressed as a ratio of RNU6B (U6); mRNA levels are expressed as a ratio to β-actin normalized to control or 5 mmol/L (5 mM) glucose.
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Figure 4: miR-146a levels are regulated with p300. In the endothelial cells, 25 mmol/L (25 mM) glucose-induced p300 mRNA upregulation (A) and increased histone acetylation (B) were prevented by p300 shRNA (p300sh) transfection. In the endothelial cells, p300 shRNA also prevented glucose-induced reduced miR-146a production (C) and FN mRNA (D) and FN protein upregulation (E) in the HUVECs. In parallel, diabetes-induced retinal p300 upregulation (G), miR-146a downregulation (H), and FN upregulation (I) were prevented by intravitreal p300 siRNA (p300si) injection. F: No significant differences were seen with respect to p300 mRNA expression when cells in 25 mmol/L glucose were transfected with miR-146a mimic or scrambled (S) mimic. *Significantly different from 5 mmol/L glucose or control (Co); **significantly different from 25 mmol/L glucose or diabetes (Di). miRNA levels are expressed as a ratio of RNU6B (U6); mRNA levels are expressed as a ratio to β-actin normalized to control or 5 mmol/L (5 mM) glucose.

Mentions: We previously have demonstrated that p300-mediated histone acetylation is a key event causing transcription of multiple proteins, including FN, in response to hyperglycemia in the endothelial cells (3,10). Furthermore, epigenetic phenomena, such as acetylation, are thought to play a significant role in the regulation of miRNA production (12). Hence, we explored the relationship of p300 in this scenario. In keeping with our previous data, in endothelial cells 25 mmol/L glucose caused significant upregulation of p300 mRNA and acetylation of histone proteins (Fig. 4A and B). Transfection of p300 shRNA significantly prevented miR-146a downregulation and FN overexpression (Fig. 4C and D). Likewise, we examined retinal tissues from diabetic and age- and sex-matched control rats. Diabetes caused increased retinal FN and p300 mRNA expression. Intravitreal p300 siRNA injection led to reduced p300 mRNA and restoration of miR-146a levels, reduction of diabetes-induced increased FN expression (Fig. 4E–G). On the other hand, miR-146a mimic transfection did not prevent glucose-induced p300 overexpression (Fig. 4H).


miR-146a-Mediated extracellular matrix protein production in chronic diabetes complications.

Feng B, Chen S, McArthur K, Wu Y, Sen S, Ding Q, Feldman RD, Chakrabarti S - Diabetes (2011)

miR-146a levels are regulated with p300. In the endothelial cells, 25 mmol/L (25 mM) glucose-induced p300 mRNA upregulation (A) and increased histone acetylation (B) were prevented by p300 shRNA (p300sh) transfection. In the endothelial cells, p300 shRNA also prevented glucose-induced reduced miR-146a production (C) and FN mRNA (D) and FN protein upregulation (E) in the HUVECs. In parallel, diabetes-induced retinal p300 upregulation (G), miR-146a downregulation (H), and FN upregulation (I) were prevented by intravitreal p300 siRNA (p300si) injection. F: No significant differences were seen with respect to p300 mRNA expression when cells in 25 mmol/L glucose were transfected with miR-146a mimic or scrambled (S) mimic. *Significantly different from 5 mmol/L glucose or control (Co); **significantly different from 25 mmol/L glucose or diabetes (Di). miRNA levels are expressed as a ratio of RNU6B (U6); mRNA levels are expressed as a ratio to β-actin normalized to control or 5 mmol/L (5 mM) glucose.
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Figure 4: miR-146a levels are regulated with p300. In the endothelial cells, 25 mmol/L (25 mM) glucose-induced p300 mRNA upregulation (A) and increased histone acetylation (B) were prevented by p300 shRNA (p300sh) transfection. In the endothelial cells, p300 shRNA also prevented glucose-induced reduced miR-146a production (C) and FN mRNA (D) and FN protein upregulation (E) in the HUVECs. In parallel, diabetes-induced retinal p300 upregulation (G), miR-146a downregulation (H), and FN upregulation (I) were prevented by intravitreal p300 siRNA (p300si) injection. F: No significant differences were seen with respect to p300 mRNA expression when cells in 25 mmol/L glucose were transfected with miR-146a mimic or scrambled (S) mimic. *Significantly different from 5 mmol/L glucose or control (Co); **significantly different from 25 mmol/L glucose or diabetes (Di). miRNA levels are expressed as a ratio of RNU6B (U6); mRNA levels are expressed as a ratio to β-actin normalized to control or 5 mmol/L (5 mM) glucose.
Mentions: We previously have demonstrated that p300-mediated histone acetylation is a key event causing transcription of multiple proteins, including FN, in response to hyperglycemia in the endothelial cells (3,10). Furthermore, epigenetic phenomena, such as acetylation, are thought to play a significant role in the regulation of miRNA production (12). Hence, we explored the relationship of p300 in this scenario. In keeping with our previous data, in endothelial cells 25 mmol/L glucose caused significant upregulation of p300 mRNA and acetylation of histone proteins (Fig. 4A and B). Transfection of p300 shRNA significantly prevented miR-146a downregulation and FN overexpression (Fig. 4C and D). Likewise, we examined retinal tissues from diabetic and age- and sex-matched control rats. Diabetes caused increased retinal FN and p300 mRNA expression. Intravitreal p300 siRNA injection led to reduced p300 mRNA and restoration of miR-146a levels, reduction of diabetes-induced increased FN expression (Fig. 4E–G). On the other hand, miR-146a mimic transfection did not prevent glucose-induced p300 overexpression (Fig. 4H).

Bottom Line: Cardiac and renal tissues were analyzed from type 1 and type 2 diabetic animals.Additional experiments showed that p300 regulates miR-146a.Similar changes were seen in the retinas, kidneys, and hearts in type 1 and type 2 diabetic animals.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Schulich School of Medicine and Dentistry and the University of Western Ontario, London, Ontario, Canada.

ABSTRACT

Objective: MicroRNAs (miRNAs), through transcriptional regulation, modulate several cellular processes. In diabetes, increased extracellular matrix protein fibronectin (FN) production is known to occur through histone acetylator p300. Here, we investigated the role of miR-146a, an FN-targeting miRNA, on FN production in diabetes and its relationship with p300.

Research design and methods: miR-146a expressions were measured in endothelial cells from large vessels and retinal microvessels in various glucose levels. FN messenger RNA expression and protein levels with or without miR-146a mimic or antagomir transfection were examined. A luciferase assay was performed to detect miR-146a's binding to FN 3'-untranslated region (UTR). Likewise, retinas from type 1 diabetic rats were studied with or without an intravitreal injection of miR-146a mimic. In situ hybridization was used to localize retinal miR-146a. Cardiac and renal tissues were analyzed from type 1 and type 2 diabetic animals.

Results: A total of 25 mmol/L glucose decreased miR-146a expression and increased FN expression compared with 5 mmol/L glucose in both cell types. miR-146a mimic transfection prevented such change, whereas miR-146a antagomir transfection in the cells in 5 mmol/L glucose caused FN upregulation. A luciferase assay confirmed miR-146a's binding to FN 3'-UTR. miR-146a was localized in the retinal endothelial cells and was decreased in diabetes. Intravitreal miR-146a mimic injection restored retinal miR-146a and decreased FN in diabetes. Additional experiments showed that p300 regulates miR-146a. Similar changes were seen in the retinas, kidneys, and hearts in type 1 and type 2 diabetic animals.

Conclusions: These studies showed a novel, glucose-induced molecular mechanism in which miR-146a participates in the transcriptional circuitry regulating extracellular matrix protein production in diabetes.

Show MeSH
Related in: MedlinePlus