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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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Related in: MedlinePlus

Endothelial cells (HUVECs) exposed to 25 mmol/L (25 mM) glucose (compared with 5 mmol/L [5 mM] glucose) showed increased FN mRNA (A) and reduced miR-146a expression (B). Such changes were not seen when the cells were incubated with 25 mmol/L l-glucose (osmotic control [Osm]). Transfection of endothelial cells with miR-146a mimics (but not the scrambled mimics) reduced basal FN expression and normalized glucose-induced upregulation of FN mRNA (A) and FN protein (C). A: The glucose-like effect (FN upregulation) was further seen when cells in 5 mmol/L glucose were transfected with miR-146a antagomir. B: The efficiency of miR-146a mimic transfection by increased miR-146a expression following miR-146a mimic transfection compared with scrambled mimics. Likewise, BRMECs showed glucose-induced miR-146a downregulation (D) and FN upregulation (E). E: Transfection of BRMECs with miR-146a mimics reduced basal FN expression and also normalized HG-induced upregulation of FN mRNA. D: The efficiency of miR-146a mimic transfection by increased miR-146a expression following miR-146a mimic transfection compared with scrambled mimics. 146a, miR-146a mimic; 146a(A), miR-146a antagomir; S, scrambled miRNA. *Significantly different from 5 mmol/L glucose; **significantly different from 25 mmol/L glucose, miRNA levels are expressed as a ratio of RNU6B (U6); mRNA levels are expressed as a ratio to β-actin and normalized to 5 mmol/L glucose.
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Figure 1: Endothelial cells (HUVECs) exposed to 25 mmol/L (25 mM) glucose (compared with 5 mmol/L [5 mM] glucose) showed increased FN mRNA (A) and reduced miR-146a expression (B). Such changes were not seen when the cells were incubated with 25 mmol/L l-glucose (osmotic control [Osm]). Transfection of endothelial cells with miR-146a mimics (but not the scrambled mimics) reduced basal FN expression and normalized glucose-induced upregulation of FN mRNA (A) and FN protein (C). A: The glucose-like effect (FN upregulation) was further seen when cells in 5 mmol/L glucose were transfected with miR-146a antagomir. B: The efficiency of miR-146a mimic transfection by increased miR-146a expression following miR-146a mimic transfection compared with scrambled mimics. Likewise, BRMECs showed glucose-induced miR-146a downregulation (D) and FN upregulation (E). E: Transfection of BRMECs with miR-146a mimics reduced basal FN expression and also normalized HG-induced upregulation of FN mRNA. D: The efficiency of miR-146a mimic transfection by increased miR-146a expression following miR-146a mimic transfection compared with scrambled mimics. 146a, miR-146a mimic; 146a(A), miR-146a antagomir; S, scrambled miRNA. *Significantly different from 5 mmol/L glucose; **significantly different from 25 mmol/L glucose, miRNA levels are expressed as a ratio of RNU6B (U6); mRNA levels are expressed as a ratio to β-actin and normalized to 5 mmol/L glucose.

Mentions: Based on the hypothesis that glucose-induced augmented FN production may, in part, be regulated by miR-146a, we investigated HUVECs exposed to 5 and 25 mmol/L glucose for 24 h. These concentrations are based on our previous dose-dependent analysis of FN mRNA and protein expression (1,5,8). We confirmed the upregulation of FN mRNA and protein following incubation with 25 mmol/L d-glucose but not by 25 mmol/L l-glucose (osmotic control) (Fig. 1A). We then extracted miRNA from these cells. Quantitative RT-PCR analysis of the endothelial cells in 25 mmol/L glucose confirmed significant downregulation of miR-146a compared with 5 mmol/L glucose. No effects were seen after incubation with 25 mmol/L l-glucose (Fig. 1B).


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)

Endothelial cells (HUVECs) exposed to 25 mmol/L (25 mM) glucose (compared with 5 mmol/L [5 mM] glucose) showed increased FN mRNA (A) and reduced miR-146a expression (B). Such changes were not seen when the cells were incubated with 25 mmol/L l-glucose (osmotic control [Osm]). Transfection of endothelial cells with miR-146a mimics (but not the scrambled mimics) reduced basal FN expression and normalized glucose-induced upregulation of FN mRNA (A) and FN protein (C). A: The glucose-like effect (FN upregulation) was further seen when cells in 5 mmol/L glucose were transfected with miR-146a antagomir. B: The efficiency of miR-146a mimic transfection by increased miR-146a expression following miR-146a mimic transfection compared with scrambled mimics. Likewise, BRMECs showed glucose-induced miR-146a downregulation (D) and FN upregulation (E). E: Transfection of BRMECs with miR-146a mimics reduced basal FN expression and also normalized HG-induced upregulation of FN mRNA. D: The efficiency of miR-146a mimic transfection by increased miR-146a expression following miR-146a mimic transfection compared with scrambled mimics. 146a, miR-146a mimic; 146a(A), miR-146a antagomir; S, scrambled miRNA. *Significantly different from 5 mmol/L glucose; **significantly different from 25 mmol/L glucose, miRNA levels are expressed as a ratio of RNU6B (U6); mRNA levels are expressed as a ratio to β-actin and normalized to 5 mmol/L glucose.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198068&req=5

Figure 1: Endothelial cells (HUVECs) exposed to 25 mmol/L (25 mM) glucose (compared with 5 mmol/L [5 mM] glucose) showed increased FN mRNA (A) and reduced miR-146a expression (B). Such changes were not seen when the cells were incubated with 25 mmol/L l-glucose (osmotic control [Osm]). Transfection of endothelial cells with miR-146a mimics (but not the scrambled mimics) reduced basal FN expression and normalized glucose-induced upregulation of FN mRNA (A) and FN protein (C). A: The glucose-like effect (FN upregulation) was further seen when cells in 5 mmol/L glucose were transfected with miR-146a antagomir. B: The efficiency of miR-146a mimic transfection by increased miR-146a expression following miR-146a mimic transfection compared with scrambled mimics. Likewise, BRMECs showed glucose-induced miR-146a downregulation (D) and FN upregulation (E). E: Transfection of BRMECs with miR-146a mimics reduced basal FN expression and also normalized HG-induced upregulation of FN mRNA. D: The efficiency of miR-146a mimic transfection by increased miR-146a expression following miR-146a mimic transfection compared with scrambled mimics. 146a, miR-146a mimic; 146a(A), miR-146a antagomir; S, scrambled miRNA. *Significantly different from 5 mmol/L glucose; **significantly different from 25 mmol/L glucose, miRNA levels are expressed as a ratio of RNU6B (U6); mRNA levels are expressed as a ratio to β-actin and normalized to 5 mmol/L glucose.
Mentions: Based on the hypothesis that glucose-induced augmented FN production may, in part, be regulated by miR-146a, we investigated HUVECs exposed to 5 and 25 mmol/L glucose for 24 h. These concentrations are based on our previous dose-dependent analysis of FN mRNA and protein expression (1,5,8). We confirmed the upregulation of FN mRNA and protein following incubation with 25 mmol/L d-glucose but not by 25 mmol/L l-glucose (osmotic control) (Fig. 1A). We then extracted miRNA from these cells. Quantitative RT-PCR analysis of the endothelial cells in 25 mmol/L glucose confirmed significant downregulation of miR-146a compared with 5 mmol/L glucose. No effects were seen after incubation with 25 mmol/L l-glucose (Fig. 1B).

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