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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 regulates IL-6 and MCP-1 expression in human cardiac cells. Relative quantification of IL-6, MCP-1 and TNF-α mRNA levels in non-differentiated human cardiac AC16 cells transfected with: (A) lacZ- or miR-146a-carrying plasmids, or (B) a human anti-miR-146a inhibitor or an anti-miR negative control (Anti-miR). The graphs represent the quantification of 18S-normalized mRNA levels, expressed as a percentage of control samples ±s.d. (A) *P<0.05 and **P<0.01 vs lacZ; (B) *P<0.05 and ***P<0.001 vs Anti-miR–TNF-α; †P<0.05 vs Anti-miR+TNF-α. (C) EMSA assay showing NF-κB DNA-binding activity after transfection of AC16 cells as described in panels A and B. Ab, antibody; Ctrl, control; NE, nuclear extracts.
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DMM020768F4: miR-146a regulates IL-6 and MCP-1 expression in human cardiac cells. Relative quantification of IL-6, MCP-1 and TNF-α mRNA levels in non-differentiated human cardiac AC16 cells transfected with: (A) lacZ- or miR-146a-carrying plasmids, or (B) a human anti-miR-146a inhibitor or an anti-miR negative control (Anti-miR). The graphs represent the quantification of 18S-normalized mRNA levels, expressed as a percentage of control samples ±s.d. (A) *P<0.05 and **P<0.01 vs lacZ; (B) *P<0.05 and ***P<0.001 vs Anti-miR–TNF-α; †P<0.05 vs Anti-miR+TNF-α. (C) EMSA assay showing NF-κB DNA-binding activity after transfection of AC16 cells as described in panels A and B. Ab, antibody; Ctrl, control; NE, nuclear extracts.

Mentions: On the other hand, miR-146a has been shown to modulate NF-κB activity through the direct targeting of well-established mediators of its activation, including interleukin-1 receptor-associated kinase (IRAK)1, IRAK2, TNF receptor-associated factor (TRAF)2 and TRAF6 (Wang et al., 2013; Tanic et al., 2012). However, none of the transcript levels for these genes was found to be modified after miR-146a overexpression or inhibition, thus indicating that they were not regulated by this miRNA in human cardiac AC16 cells (see supplementary material Fig. S3). However, miR-146a overexpression inhibited IL-6 (∼40% reduction, P<0.05 vs lacZ) and MCP-1 (∼50% reduction, P<0.01) transcript levels, although TNF-α levels remained unaltered (Fig. 4A). In consonance with this, transfection of AC16 cells with the anti-miR-146a inhibitor upregulated the expression of IL-6 (1.3-fold, P<0.05 vs anti-miR+TNF-α), although only when the pro-inflammatory stimulus TNF-α was added to the medium (Fig. 4B). As previously reported (Palomer et al., 2009), treatment of AC16 cells with TNF-α (100 ng/ml for 24 h) significantly induced the expression of IL-6, MCP-1 and TNF-α, regardless of miR-146a levels. After that, an EMSA was carried out to verify whether modulation of miR-146a levels in AC16 cells led to changes in NF-κB activity. As shown in Fig. 4C, NF-κB formed four specific DNA-binding complexes (I to IV) with nuclear proteins. The competitor lane confirmed that all four complexes were specific for the NF-κB probe, and supershift analyses provided evidence that only complexes I and II contained the p65 subunit of NF-κB. Interestingly, complexes I, II and III were increased in TNF-α-treated cells, but no variations were detected after miR-146a modulation. These results suggest that IL-6 and MCP-1 gene expression might be regulated by transcription factors other than NF-κB.Fig. 4.


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 regulates IL-6 and MCP-1 expression in human cardiac cells. Relative quantification of IL-6, MCP-1 and TNF-α mRNA levels in non-differentiated human cardiac AC16 cells transfected with: (A) lacZ- or miR-146a-carrying plasmids, or (B) a human anti-miR-146a inhibitor or an anti-miR negative control (Anti-miR). The graphs represent the quantification of 18S-normalized mRNA levels, expressed as a percentage of control samples ±s.d. (A) *P<0.05 and **P<0.01 vs lacZ; (B) *P<0.05 and ***P<0.001 vs Anti-miR–TNF-α; †P<0.05 vs Anti-miR+TNF-α. (C) EMSA assay showing NF-κB DNA-binding activity after transfection of AC16 cells as described in panels A and B. Ab, antibody; Ctrl, control; NE, nuclear extracts.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4582106&req=5

DMM020768F4: miR-146a regulates IL-6 and MCP-1 expression in human cardiac cells. Relative quantification of IL-6, MCP-1 and TNF-α mRNA levels in non-differentiated human cardiac AC16 cells transfected with: (A) lacZ- or miR-146a-carrying plasmids, or (B) a human anti-miR-146a inhibitor or an anti-miR negative control (Anti-miR). The graphs represent the quantification of 18S-normalized mRNA levels, expressed as a percentage of control samples ±s.d. (A) *P<0.05 and **P<0.01 vs lacZ; (B) *P<0.05 and ***P<0.001 vs Anti-miR–TNF-α; †P<0.05 vs Anti-miR+TNF-α. (C) EMSA assay showing NF-κB DNA-binding activity after transfection of AC16 cells as described in panels A and B. Ab, antibody; Ctrl, control; NE, nuclear extracts.
Mentions: On the other hand, miR-146a has been shown to modulate NF-κB activity through the direct targeting of well-established mediators of its activation, including interleukin-1 receptor-associated kinase (IRAK)1, IRAK2, TNF receptor-associated factor (TRAF)2 and TRAF6 (Wang et al., 2013; Tanic et al., 2012). However, none of the transcript levels for these genes was found to be modified after miR-146a overexpression or inhibition, thus indicating that they were not regulated by this miRNA in human cardiac AC16 cells (see supplementary material Fig. S3). However, miR-146a overexpression inhibited IL-6 (∼40% reduction, P<0.05 vs lacZ) and MCP-1 (∼50% reduction, P<0.01) transcript levels, although TNF-α levels remained unaltered (Fig. 4A). In consonance with this, transfection of AC16 cells with the anti-miR-146a inhibitor upregulated the expression of IL-6 (1.3-fold, P<0.05 vs anti-miR+TNF-α), although only when the pro-inflammatory stimulus TNF-α was added to the medium (Fig. 4B). As previously reported (Palomer et al., 2009), treatment of AC16 cells with TNF-α (100 ng/ml for 24 h) significantly induced the expression of IL-6, MCP-1 and TNF-α, regardless of miR-146a levels. After that, an EMSA was carried out to verify whether modulation of miR-146a levels in AC16 cells led to changes in NF-κB activity. As shown in Fig. 4C, NF-κB formed four specific DNA-binding complexes (I to IV) with nuclear proteins. The competitor lane confirmed that all four complexes were specific for the NF-κB probe, and supershift analyses provided evidence that only complexes I and II contained the p65 subunit of NF-κB. Interestingly, complexes I, II and III were increased in TNF-α-treated cells, but no variations were detected after miR-146a modulation. These results suggest that IL-6 and MCP-1 gene expression might be regulated by transcription factors other than NF-κB.Fig. 4.

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