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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

miR-146a–mediated alteration of retinal FN. A: Poorly controlled diabetes caused a reduction of rat retinal miR-146a levels. B and C: Representative LNA-ISH study of retinal tissues in a control (Co) and a diabetic (Di) rat retina showing localization of miR-146a (blue chromogen) in the retinal capillaries (arrow) and in the cells of inner nuclear layer (large arrow), possibly both in the glial and neuronal elements. Insets show an enlarged view of the capillary with miR-146a localization (arrow) in a control rat and reduced level of endothelial miR-146a in the retina of a diabetic rat (arrow). Diabetes-induced augmented FN mRNA (C) and protein levels (D) (as measured by ELISA) in the rat retina can be prevented by intravitreal miR-146a mimic (but not by scrambled [S] mimics) injection. E: Efficiency of intravitreal delivery as demonstrated by increased retinal miR-146a expression after an intravitreal injection of miR-146a mimic compared with scrambled mimic. *Significantly different from control; **significantly different from diabetic or diabetic plus scrambled. miRNA levels are expressed as a ratio of RNU6B (U6) normalized to control or diabetic plus scrambled (E). mRNA levels are expressed as a ratio to β-actin and normalized to control. Alkaline phosphatase was used as chromogen (blue) with no counterstain in LNA-ISH. R, red blood cell. (A high-quality digital representation of this figure is available in the online issue.)
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Figure 3: miR-146a–mediated alteration of retinal FN. A: Poorly controlled diabetes caused a reduction of rat retinal miR-146a levels. B and C: Representative LNA-ISH study of retinal tissues in a control (Co) and a diabetic (Di) rat retina showing localization of miR-146a (blue chromogen) in the retinal capillaries (arrow) and in the cells of inner nuclear layer (large arrow), possibly both in the glial and neuronal elements. Insets show an enlarged view of the capillary with miR-146a localization (arrow) in a control rat and reduced level of endothelial miR-146a in the retina of a diabetic rat (arrow). Diabetes-induced augmented FN mRNA (C) and protein levels (D) (as measured by ELISA) in the rat retina can be prevented by intravitreal miR-146a mimic (but not by scrambled [S] mimics) injection. E: Efficiency of intravitreal delivery as demonstrated by increased retinal miR-146a expression after an intravitreal injection of miR-146a mimic compared with scrambled mimic. *Significantly different from control; **significantly different from diabetic or diabetic plus scrambled. miRNA levels are expressed as a ratio of RNU6B (U6) normalized to control or diabetic plus scrambled (E). mRNA levels are expressed as a ratio to β-actin and normalized to control. Alkaline phosphatase was used as chromogen (blue) with no counterstain in LNA-ISH. R, red blood cell. (A high-quality digital representation of this figure is available in the online issue.)

Mentions: After establishing that miR-146a regulated FN in HUVECs and in BRMECs, we expanded our study to investigate whether the mechanisms seen in these cells were important in the development of retinal microangiopathy in a well-established animal model. STZ-induced diabetic rats showed hyperglycemia (serum glucose of diabetes 23.5 ± 3.7 mmol/L vs. controls 5.3 ± 0.4 mmol/L, P < 0.001) and reduced body weight (body weight of diabetes 304.2 ± 58.8 g vs. controls 400.7 ± 32.4 g, P < 0.001). We initially performed real-time PCR analysis of the retinal tissue from rats after 1 month of diabetes. We selected this time point because we have previously shown that diabetes-induced increased ECM protein and vasoactive factor expression is established at this time point (2,3). Real-time PCR analysis demonstrated that 1 month of poorly controlled diabetes causes significant downregulation of miR-146a (Fig. 3A). We further confirmed these results using in situ hybridization and LNA probes. miR-146a was localized in the neuronal, glial, and endothelial cells in the retina. Overall expression was reduced in the retina in diabetes, including that in the endothelial cells (Fig. 3B).


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–mediated alteration of retinal FN. A: Poorly controlled diabetes caused a reduction of rat retinal miR-146a levels. B and C: Representative LNA-ISH study of retinal tissues in a control (Co) and a diabetic (Di) rat retina showing localization of miR-146a (blue chromogen) in the retinal capillaries (arrow) and in the cells of inner nuclear layer (large arrow), possibly both in the glial and neuronal elements. Insets show an enlarged view of the capillary with miR-146a localization (arrow) in a control rat and reduced level of endothelial miR-146a in the retina of a diabetic rat (arrow). Diabetes-induced augmented FN mRNA (C) and protein levels (D) (as measured by ELISA) in the rat retina can be prevented by intravitreal miR-146a mimic (but not by scrambled [S] mimics) injection. E: Efficiency of intravitreal delivery as demonstrated by increased retinal miR-146a expression after an intravitreal injection of miR-146a mimic compared with scrambled mimic. *Significantly different from control; **significantly different from diabetic or diabetic plus scrambled. miRNA levels are expressed as a ratio of RNU6B (U6) normalized to control or diabetic plus scrambled (E). mRNA levels are expressed as a ratio to β-actin and normalized to control. Alkaline phosphatase was used as chromogen (blue) with no counterstain in LNA-ISH. R, red blood cell. (A high-quality digital representation of this figure is available in the online issue.)
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Figure 3: miR-146a–mediated alteration of retinal FN. A: Poorly controlled diabetes caused a reduction of rat retinal miR-146a levels. B and C: Representative LNA-ISH study of retinal tissues in a control (Co) and a diabetic (Di) rat retina showing localization of miR-146a (blue chromogen) in the retinal capillaries (arrow) and in the cells of inner nuclear layer (large arrow), possibly both in the glial and neuronal elements. Insets show an enlarged view of the capillary with miR-146a localization (arrow) in a control rat and reduced level of endothelial miR-146a in the retina of a diabetic rat (arrow). Diabetes-induced augmented FN mRNA (C) and protein levels (D) (as measured by ELISA) in the rat retina can be prevented by intravitreal miR-146a mimic (but not by scrambled [S] mimics) injection. E: Efficiency of intravitreal delivery as demonstrated by increased retinal miR-146a expression after an intravitreal injection of miR-146a mimic compared with scrambled mimic. *Significantly different from control; **significantly different from diabetic or diabetic plus scrambled. miRNA levels are expressed as a ratio of RNU6B (U6) normalized to control or diabetic plus scrambled (E). mRNA levels are expressed as a ratio to β-actin and normalized to control. Alkaline phosphatase was used as chromogen (blue) with no counterstain in LNA-ISH. R, red blood cell. (A high-quality digital representation of this figure is available in the online issue.)
Mentions: After establishing that miR-146a regulated FN in HUVECs and in BRMECs, we expanded our study to investigate whether the mechanisms seen in these cells were important in the development of retinal microangiopathy in a well-established animal model. STZ-induced diabetic rats showed hyperglycemia (serum glucose of diabetes 23.5 ± 3.7 mmol/L vs. controls 5.3 ± 0.4 mmol/L, P < 0.001) and reduced body weight (body weight of diabetes 304.2 ± 58.8 g vs. controls 400.7 ± 32.4 g, P < 0.001). We initially performed real-time PCR analysis of the retinal tissue from rats after 1 month of diabetes. We selected this time point because we have previously shown that diabetes-induced increased ECM protein and vasoactive factor expression is established at this time point (2,3). Real-time PCR analysis demonstrated that 1 month of poorly controlled diabetes causes significant downregulation of miR-146a (Fig. 3A). We further confirmed these results using in situ hybridization and LNA probes. miR-146a was localized in the neuronal, glial, and endothelial cells in the retina. Overall expression was reduced in the retina in diabetes, including that in the endothelial cells (Fig. 3B).

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