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miR-146a targets Fos expression in human cardiac cells.

Palomer X, Capdevila-Busquets E, Botteri G, Davidson MM, Rodríguez C, Martínez-González J, Vidal F, Barroso E, Chan TO, Feldman AM, Vázquez-Carrera M - Dis Model Mech (2015)

Bottom Line: These changes correlated with a diminution in the DNA-binding activity of AP-1, the Fos-containing transcription factor complex.The specific regulation of this MMP by miR-146a was further confirmed at the secretion and enzymatic activity levels, as well as after anti-miR-mediated miR-146a inhibition.The results reported here demonstrate that Fos is a direct target of miR-146a activity and that downregulation of the Fos-AP-1 pathway by miR-146a has the capacity to inhibit MMP-9 activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Therapeutic Chemistry, IBUB (Institut de Biomedicina de la Universitat de Barcelona) and CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Faculty of Pharmacy, University of Barcelona, Diagonal 643, Barcelona E-08028, Spain.

No MeSH data available.


Related in: MedlinePlus

miR-146a overexpression inhibits Fos in human cardiac cells. Relative quantification of miR-146a (A) and Fos (B) mRNA levels in human cardiac AC16 cells transfected with lacZ- or miR-146a-carrying plasmids. The graph represents the quantification of (A) U6sRNA- or (B) 18S-normalized mRNA levels, expressed as a percentage of control samples ±s.d. *P<0.05 and **P<0.01 vs lacZ. (C) Western-blot analysis showing the protein levels of Fos, Jun and p65 in cytosolic (CP) and nuclear (NE) protein fractions obtained from human cardiac AC16 cells as described in panel A. To show equal loading of protein, the actin and lamin B signals from the same blot are included. The graphs at the bottom of panel C represent the quantification of protein levels normalized to actin (CP) or lamin B (NE), expressed as a percentage of CP or NE control samples ±s.d. The blot data are representative of two separate experiments. *P<0.05 vs lacZ CP; †P<0.05 vs lacZ NE. (D) EMSA assay showing AP-1 DNA-binding activity after transfection of AC16 cells as described in panel A. Ab, antibody; NE, nuclear extracts.
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DMM020768F2: miR-146a overexpression inhibits Fos in human cardiac cells. Relative quantification of miR-146a (A) and Fos (B) mRNA levels in human cardiac AC16 cells transfected with lacZ- or miR-146a-carrying plasmids. The graph represents the quantification of (A) U6sRNA- or (B) 18S-normalized mRNA levels, expressed as a percentage of control samples ±s.d. *P<0.05 and **P<0.01 vs lacZ. (C) Western-blot analysis showing the protein levels of Fos, Jun and p65 in cytosolic (CP) and nuclear (NE) protein fractions obtained from human cardiac AC16 cells as described in panel A. To show equal loading of protein, the actin and lamin B signals from the same blot are included. The graphs at the bottom of panel C represent the quantification of protein levels normalized to actin (CP) or lamin B (NE), expressed as a percentage of CP or NE control samples ±s.d. The blot data are representative of two separate experiments. *P<0.05 vs lacZ CP; †P<0.05 vs lacZ NE. (D) EMSA assay showing AP-1 DNA-binding activity after transfection of AC16 cells as described in panel A. Ab, antibody; NE, nuclear extracts.

Mentions: To determine whether Fos was a direct target of miR-146a activity, human AC16 cardiac cells were transfected with a plasmid carrying pre-miR-146a in the absence of TNF-α, which yielded an important increase in miR-146a expression (15-fold, P<0.05 vs lacZ, Fig. 2A). Compatible with our hypothesis, real-time RT-PCR analysis revealed that this miRNA was inhibiting Fos expression (∼45% reduction, P<0.01, Fig. 2B). Such inhibition resulted in a significant downregulation of Fos protein levels at both the cytoplasm (CP fraction, ∼50% reduction, P<0.05) and the nucleus (NE fraction, ∼50% reduction, P<0.05) of miR-146a-transfected cells (Fig. 2C), and caused a significant decrease in the DNA-binding activity of the AP-1 transcription factor (Fig. 2D). In agreement with this, downregulation of miR-146a levels (∼98% reduction, Fig. 3A) by transfecting AC16 cells with a human anti-miR-146a inhibitor coincided with a significant increase in Fos expression (1.9-fold, P<0.001 vs Ctrl anti-miR, Fig. 3B). In addition, the anti-miR-146a inhibitor partially reversed the effects of TNF-α on Fos expression (1.7-fold, P<0.001 vs anti-miR+TNF-α). In agreement with gene expression data, Fos protein levels were enhanced by 1.4-fold (P<0.05 vs Ctrl anti-miR+TNF-α) in the nucleus of AC16 cells transfected with anti-miR-146a (NE fraction, Fig. 3C). In order to demonstrate the specificity of Fos regulation by miR-146a, the protein levels of Jun and the p65 subunit of NF-κB were also determined in cytoplasmic and nuclear extracts of cells overexpressing miR-146a (Fig. 2C) and after downregulation of miR-146a with an anti-miR-146a inhibitor (Fig. 3C). It is worth mentioning that p65 and Jun were not altered after modulation of miR-146a levels. As expected, p65 protein levels were increased in the nucleus after TNF-α treatment, owing to NF-κB activation. In non-stimulated cells, NF-κB remains inactive in the cytoplasm owing to its heterodimerization with the inhibitor protein IκB. In the presence of a stimulus (e.g. TNF-α), the IκB kinase (IKK) complex phosphorylates IκB, which induces ubiquitylation and ensuing proteasome-mediated degradation of the latter. The subsequent release of the NF-κB heterodimer makes possible its translocation to the nucleus, where it can begin the transcription of its target genes.Fig. 2.


miR-146a targets Fos expression in human cardiac cells.

Palomer X, Capdevila-Busquets E, Botteri G, Davidson MM, Rodríguez C, Martínez-González J, Vidal F, Barroso E, Chan TO, Feldman AM, Vázquez-Carrera M - Dis Model Mech (2015)

miR-146a overexpression inhibits Fos in human cardiac cells. Relative quantification of miR-146a (A) and Fos (B) mRNA levels in human cardiac AC16 cells transfected with lacZ- or miR-146a-carrying plasmids. The graph represents the quantification of (A) U6sRNA- or (B) 18S-normalized mRNA levels, expressed as a percentage of control samples ±s.d. *P<0.05 and **P<0.01 vs lacZ. (C) Western-blot analysis showing the protein levels of Fos, Jun and p65 in cytosolic (CP) and nuclear (NE) protein fractions obtained from human cardiac AC16 cells as described in panel A. To show equal loading of protein, the actin and lamin B signals from the same blot are included. The graphs at the bottom of panel C represent the quantification of protein levels normalized to actin (CP) or lamin B (NE), expressed as a percentage of CP or NE control samples ±s.d. The blot data are representative of two separate experiments. *P<0.05 vs lacZ CP; †P<0.05 vs lacZ NE. (D) EMSA assay showing AP-1 DNA-binding activity after transfection of AC16 cells as described in panel A. Ab, antibody; NE, nuclear extracts.
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DMM020768F2: miR-146a overexpression inhibits Fos in human cardiac cells. Relative quantification of miR-146a (A) and Fos (B) mRNA levels in human cardiac AC16 cells transfected with lacZ- or miR-146a-carrying plasmids. The graph represents the quantification of (A) U6sRNA- or (B) 18S-normalized mRNA levels, expressed as a percentage of control samples ±s.d. *P<0.05 and **P<0.01 vs lacZ. (C) Western-blot analysis showing the protein levels of Fos, Jun and p65 in cytosolic (CP) and nuclear (NE) protein fractions obtained from human cardiac AC16 cells as described in panel A. To show equal loading of protein, the actin and lamin B signals from the same blot are included. The graphs at the bottom of panel C represent the quantification of protein levels normalized to actin (CP) or lamin B (NE), expressed as a percentage of CP or NE control samples ±s.d. The blot data are representative of two separate experiments. *P<0.05 vs lacZ CP; †P<0.05 vs lacZ NE. (D) EMSA assay showing AP-1 DNA-binding activity after transfection of AC16 cells as described in panel A. Ab, antibody; NE, nuclear extracts.
Mentions: To determine whether Fos was a direct target of miR-146a activity, human AC16 cardiac cells were transfected with a plasmid carrying pre-miR-146a in the absence of TNF-α, which yielded an important increase in miR-146a expression (15-fold, P<0.05 vs lacZ, Fig. 2A). Compatible with our hypothesis, real-time RT-PCR analysis revealed that this miRNA was inhibiting Fos expression (∼45% reduction, P<0.01, Fig. 2B). Such inhibition resulted in a significant downregulation of Fos protein levels at both the cytoplasm (CP fraction, ∼50% reduction, P<0.05) and the nucleus (NE fraction, ∼50% reduction, P<0.05) of miR-146a-transfected cells (Fig. 2C), and caused a significant decrease in the DNA-binding activity of the AP-1 transcription factor (Fig. 2D). In agreement with this, downregulation of miR-146a levels (∼98% reduction, Fig. 3A) by transfecting AC16 cells with a human anti-miR-146a inhibitor coincided with a significant increase in Fos expression (1.9-fold, P<0.001 vs Ctrl anti-miR, Fig. 3B). In addition, the anti-miR-146a inhibitor partially reversed the effects of TNF-α on Fos expression (1.7-fold, P<0.001 vs anti-miR+TNF-α). In agreement with gene expression data, Fos protein levels were enhanced by 1.4-fold (P<0.05 vs Ctrl anti-miR+TNF-α) in the nucleus of AC16 cells transfected with anti-miR-146a (NE fraction, Fig. 3C). In order to demonstrate the specificity of Fos regulation by miR-146a, the protein levels of Jun and the p65 subunit of NF-κB were also determined in cytoplasmic and nuclear extracts of cells overexpressing miR-146a (Fig. 2C) and after downregulation of miR-146a with an anti-miR-146a inhibitor (Fig. 3C). It is worth mentioning that p65 and Jun were not altered after modulation of miR-146a levels. As expected, p65 protein levels were increased in the nucleus after TNF-α treatment, owing to NF-κB activation. In non-stimulated cells, NF-κB remains inactive in the cytoplasm owing to its heterodimerization with the inhibitor protein IκB. In the presence of a stimulus (e.g. TNF-α), the IκB kinase (IKK) complex phosphorylates IκB, which induces ubiquitylation and ensuing proteasome-mediated degradation of the latter. The subsequent release of the NF-κB heterodimer makes possible its translocation to the nucleus, where it can begin the transcription of its target genes.Fig. 2.

Bottom Line: These changes correlated with a diminution in the DNA-binding activity of AP-1, the Fos-containing transcription factor complex.The specific regulation of this MMP by miR-146a was further confirmed at the secretion and enzymatic activity levels, as well as after anti-miR-mediated miR-146a inhibition.The results reported here demonstrate that Fos is a direct target of miR-146a activity and that downregulation of the Fos-AP-1 pathway by miR-146a has the capacity to inhibit MMP-9 activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Therapeutic Chemistry, IBUB (Institut de Biomedicina de la Universitat de Barcelona) and CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Faculty of Pharmacy, University of Barcelona, Diagonal 643, Barcelona E-08028, Spain.

No MeSH data available.


Related in: MedlinePlus