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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

A: Alignment of FN 3′-UTR (and mutated FN 3′-UTR) sequence with mature miR-146a based on bioinformatics predictions (www.targetscan.org, www.microrna.org, and www.ebi.ac.uk1). The 5′ end of the mature miR-146a is the seed sequence and has perfect complementarity with seven nucleotides of the 3′-UTR of FN. In the mutated sequence (small caps identifying mutated nucleotides), such complementarity was lost. B and C: Luciferase reporter assay using rat and human FN, respectively, showing dose-dependent binding of FN 3′-UTR with miR-146a, whereas mutated (mut) FN 3′-UTR abrogated the inhibitory effects of miR-146a. Relative luciferase activities were expressed as luminescence units and normalized for β-galactosidase expression. **Significantly different from vector (V) or mut V, FN 3′-UTR luciferase plasmids plus β-galactosidase plasmids; V-mut, mutated FN 3′-UTR luciferase plasmids plus β-galactosidase plasmids. (A high-quality color representation of this figure is available in the online issue.)
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Figure 2: A: Alignment of FN 3′-UTR (and mutated FN 3′-UTR) sequence with mature miR-146a based on bioinformatics predictions (www.targetscan.org, www.microrna.org, and www.ebi.ac.uk1). The 5′ end of the mature miR-146a is the seed sequence and has perfect complementarity with seven nucleotides of the 3′-UTR of FN. In the mutated sequence (small caps identifying mutated nucleotides), such complementarity was lost. B and C: Luciferase reporter assay using rat and human FN, respectively, showing dose-dependent binding of FN 3′-UTR with miR-146a, whereas mutated (mut) FN 3′-UTR abrogated the inhibitory effects of miR-146a. Relative luciferase activities were expressed as luminescence units and normalized for β-galactosidase expression. **Significantly different from vector (V) or mut V, FN 3′-UTR luciferase plasmids plus β-galactosidase plasmids; V-mut, mutated FN 3′-UTR luciferase plasmids plus β-galactosidase plasmids. (A high-quality color representation of this figure is available in the online issue.)

Mentions: We then wanted to confirm that the effects of miR-146a are indeed mediated by direct binding of miR-146a with the 3′-UTR of the FN gene. To do this, we carried out luciferase assays. Such assays were performed in HEK293A cells. Luciferase reporter plasmids were prepared, containing cloned miR-146a binding sites for the FN 3′-UTR (of both rat and human FN in separate experiments) or mutated 3′-UTR. The plasmids were cotransfected with miR-146a mimic, causing overexpression of miR-146a. Such miR-146a overexpression significantly repressed FN 3′-UTR luciferase activity. However, no such repression was seen when we performed similar experiments using mutated 3′-UTR (Fig. 2). These data indicated that binding of miR-146a with the 3′-UTR of the FN gene was required to produce its effects.


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)

A: Alignment of FN 3′-UTR (and mutated FN 3′-UTR) sequence with mature miR-146a based on bioinformatics predictions (www.targetscan.org, www.microrna.org, and www.ebi.ac.uk1). The 5′ end of the mature miR-146a is the seed sequence and has perfect complementarity with seven nucleotides of the 3′-UTR of FN. In the mutated sequence (small caps identifying mutated nucleotides), such complementarity was lost. B and C: Luciferase reporter assay using rat and human FN, respectively, showing dose-dependent binding of FN 3′-UTR with miR-146a, whereas mutated (mut) FN 3′-UTR abrogated the inhibitory effects of miR-146a. Relative luciferase activities were expressed as luminescence units and normalized for β-galactosidase expression. **Significantly different from vector (V) or mut V, FN 3′-UTR luciferase plasmids plus β-galactosidase plasmids; V-mut, mutated FN 3′-UTR luciferase plasmids plus β-galactosidase plasmids. (A high-quality color representation of this figure is available in the online issue.)
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 2: A: Alignment of FN 3′-UTR (and mutated FN 3′-UTR) sequence with mature miR-146a based on bioinformatics predictions (www.targetscan.org, www.microrna.org, and www.ebi.ac.uk1). The 5′ end of the mature miR-146a is the seed sequence and has perfect complementarity with seven nucleotides of the 3′-UTR of FN. In the mutated sequence (small caps identifying mutated nucleotides), such complementarity was lost. B and C: Luciferase reporter assay using rat and human FN, respectively, showing dose-dependent binding of FN 3′-UTR with miR-146a, whereas mutated (mut) FN 3′-UTR abrogated the inhibitory effects of miR-146a. Relative luciferase activities were expressed as luminescence units and normalized for β-galactosidase expression. **Significantly different from vector (V) or mut V, FN 3′-UTR luciferase plasmids plus β-galactosidase plasmids; V-mut, mutated FN 3′-UTR luciferase plasmids plus β-galactosidase plasmids. (A high-quality color representation of this figure is available in the online issue.)
Mentions: We then wanted to confirm that the effects of miR-146a are indeed mediated by direct binding of miR-146a with the 3′-UTR of the FN gene. To do this, we carried out luciferase assays. Such assays were performed in HEK293A cells. Luciferase reporter plasmids were prepared, containing cloned miR-146a binding sites for the FN 3′-UTR (of both rat and human FN in separate experiments) or mutated 3′-UTR. The plasmids were cotransfected with miR-146a mimic, causing overexpression of miR-146a. Such miR-146a overexpression significantly repressed FN 3′-UTR luciferase activity. However, no such repression was seen when we performed similar experiments using mutated 3′-UTR (Fig. 2). These data indicated that binding of miR-146a with the 3′-UTR of the FN gene was required to produce its effects.

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