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miR-185 plays an anti-hypertrophic role in the heart via multiple targets in the calcium-signaling pathways.

Kim JO, Song DW, Kwon EJ, Hong SE, Song HK, Min CK, Kim do H - PLoS ONE (2015)

Bottom Line: The results showed that up-regulation of miR-185 led to anti-hypertrophic effects, while down-regulation led to pro-hypertrophic effects, suggesting that miR-185 has an anti-hypertrophic role in the heart.The expression of phospho-phospholamban (Thr-17), a marker of CaMKIIδ activity, was also significantly reduced by miR-185.In conclusion, miR-185 effectively blocked cardiac hypertrophy signaling through multiple targets, rendering it a potential drug target for diseases such as heart failure.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences and Systems Biology Research Center, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea.

ABSTRACT
MicroRNA (miRNA) is an endogenous non-coding RNA species that either inhibits RNA translation or promotes degradation of target mRNAs. miRNAs often regulate cellular signaling by targeting multiple genes within the pathways. In the present study, using Gene Set Analysis, a useful bioinformatics tool to identify miRNAs with multiple target genes in the same pathways, we identified miR-185 as a key candidate regulator of cardiac hypertrophy. Using a mouse model, we found that miR-185 was significantly down-regulated in myocardial cells during cardiac hypertrophy induced by transverse aortic constriction. To confirm that miR-185 is an anti-hypertrophic miRNA, genetic manipulation studies such as overexpression and knock-down of miR-185 in neonatal rat ventricular myocytes were conducted. The results showed that up-regulation of miR-185 led to anti-hypertrophic effects, while down-regulation led to pro-hypertrophic effects, suggesting that miR-185 has an anti-hypertrophic role in the heart. Our study further identified Camk2d, Ncx1, and Nfatc3 as direct targets of miR-185. The activity of Nuclear Factor of Activated T-cell (NFAT) and calcium/calmodulin-dependent protein kinase II delta (CaMKIIδ) was negatively regulated by miR-185 as assessed by NFAT-luciferase activity and western blotting. The expression of phospho-phospholamban (Thr-17), a marker of CaMKIIδ activity, was also significantly reduced by miR-185. In conclusion, miR-185 effectively blocked cardiac hypertrophy signaling through multiple targets, rendering it a potential drug target for diseases such as heart failure.

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miR-185 negatively regulates NFAT activity in NRVMs.(A) Schematic representation of the luciferase reporter construct driven by nine tandem NFAT binding sites. (B) Relative luciferase activity in NRVMs transfected with NC or miR-185 inhibitor. NRVMs were stimulated by ET-1 (10 nmol/L) for 24 h. pRL-TK was transfected for normalization and as an internal control for transfection efficiency. (C) 72 h after transfection of NC or miR-185 mimic, the level of p-NFATC3 and total NFATC3 were analyzed by western blotting. α-tubulin was used as a loading control. Representative western blots (left) and quantified western blots (right). The data are expressed as mean ± SEM of more than three independent experiments; *P < 0.05, **P < 0.001.
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pone.0122509.g004: miR-185 negatively regulates NFAT activity in NRVMs.(A) Schematic representation of the luciferase reporter construct driven by nine tandem NFAT binding sites. (B) Relative luciferase activity in NRVMs transfected with NC or miR-185 inhibitor. NRVMs were stimulated by ET-1 (10 nmol/L) for 24 h. pRL-TK was transfected for normalization and as an internal control for transfection efficiency. (C) 72 h after transfection of NC or miR-185 mimic, the level of p-NFATC3 and total NFATC3 were analyzed by western blotting. α-tubulin was used as a loading control. Representative western blots (left) and quantified western blots (right). The data are expressed as mean ± SEM of more than three independent experiments; *P < 0.05, **P < 0.001.

Mentions: Based on evidence concerning the regulation of calcineurin-NFAT signaling by miR-185 (Fig. 3C, Figures B and J in S1 File), we further examined whether inhibition of miR-185 could activate calcineurin-dependent NFAT transcription factor by luciferase reporter assay employing a reporter under transcriptional control of nine NFAT binding sites (Fig. 4A). Transfection of NRVMs with miR-185 inhibitor led to marked induction of the NFAT-dependent luciferase activity compared with the controls for the basal state and for the ET-1 stimulation (Fig. 4B). In contrast, overexpression of miR-185 significantly induced phosphorylation of NFATC3, while significantly decreasing the total NFATC3, as assessed by western blotting (Fig. 4C), suggesting that miR-185 negatively regulates calcineurin-NFAT signaling.


miR-185 plays an anti-hypertrophic role in the heart via multiple targets in the calcium-signaling pathways.

Kim JO, Song DW, Kwon EJ, Hong SE, Song HK, Min CK, Kim do H - PLoS ONE (2015)

miR-185 negatively regulates NFAT activity in NRVMs.(A) Schematic representation of the luciferase reporter construct driven by nine tandem NFAT binding sites. (B) Relative luciferase activity in NRVMs transfected with NC or miR-185 inhibitor. NRVMs were stimulated by ET-1 (10 nmol/L) for 24 h. pRL-TK was transfected for normalization and as an internal control for transfection efficiency. (C) 72 h after transfection of NC or miR-185 mimic, the level of p-NFATC3 and total NFATC3 were analyzed by western blotting. α-tubulin was used as a loading control. Representative western blots (left) and quantified western blots (right). The data are expressed as mean ± SEM of more than three independent experiments; *P < 0.05, **P < 0.001.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4358957&req=5

pone.0122509.g004: miR-185 negatively regulates NFAT activity in NRVMs.(A) Schematic representation of the luciferase reporter construct driven by nine tandem NFAT binding sites. (B) Relative luciferase activity in NRVMs transfected with NC or miR-185 inhibitor. NRVMs were stimulated by ET-1 (10 nmol/L) for 24 h. pRL-TK was transfected for normalization and as an internal control for transfection efficiency. (C) 72 h after transfection of NC or miR-185 mimic, the level of p-NFATC3 and total NFATC3 were analyzed by western blotting. α-tubulin was used as a loading control. Representative western blots (left) and quantified western blots (right). The data are expressed as mean ± SEM of more than three independent experiments; *P < 0.05, **P < 0.001.
Mentions: Based on evidence concerning the regulation of calcineurin-NFAT signaling by miR-185 (Fig. 3C, Figures B and J in S1 File), we further examined whether inhibition of miR-185 could activate calcineurin-dependent NFAT transcription factor by luciferase reporter assay employing a reporter under transcriptional control of nine NFAT binding sites (Fig. 4A). Transfection of NRVMs with miR-185 inhibitor led to marked induction of the NFAT-dependent luciferase activity compared with the controls for the basal state and for the ET-1 stimulation (Fig. 4B). In contrast, overexpression of miR-185 significantly induced phosphorylation of NFATC3, while significantly decreasing the total NFATC3, as assessed by western blotting (Fig. 4C), suggesting that miR-185 negatively regulates calcineurin-NFAT signaling.

Bottom Line: The results showed that up-regulation of miR-185 led to anti-hypertrophic effects, while down-regulation led to pro-hypertrophic effects, suggesting that miR-185 has an anti-hypertrophic role in the heart.The expression of phospho-phospholamban (Thr-17), a marker of CaMKIIδ activity, was also significantly reduced by miR-185.In conclusion, miR-185 effectively blocked cardiac hypertrophy signaling through multiple targets, rendering it a potential drug target for diseases such as heart failure.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences and Systems Biology Research Center, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea.

ABSTRACT
MicroRNA (miRNA) is an endogenous non-coding RNA species that either inhibits RNA translation or promotes degradation of target mRNAs. miRNAs often regulate cellular signaling by targeting multiple genes within the pathways. In the present study, using Gene Set Analysis, a useful bioinformatics tool to identify miRNAs with multiple target genes in the same pathways, we identified miR-185 as a key candidate regulator of cardiac hypertrophy. Using a mouse model, we found that miR-185 was significantly down-regulated in myocardial cells during cardiac hypertrophy induced by transverse aortic constriction. To confirm that miR-185 is an anti-hypertrophic miRNA, genetic manipulation studies such as overexpression and knock-down of miR-185 in neonatal rat ventricular myocytes were conducted. The results showed that up-regulation of miR-185 led to anti-hypertrophic effects, while down-regulation led to pro-hypertrophic effects, suggesting that miR-185 has an anti-hypertrophic role in the heart. Our study further identified Camk2d, Ncx1, and Nfatc3 as direct targets of miR-185. The activity of Nuclear Factor of Activated T-cell (NFAT) and calcium/calmodulin-dependent protein kinase II delta (CaMKIIδ) was negatively regulated by miR-185 as assessed by NFAT-luciferase activity and western blotting. The expression of phospho-phospholamban (Thr-17), a marker of CaMKIIδ activity, was also significantly reduced by miR-185. In conclusion, miR-185 effectively blocked cardiac hypertrophy signaling through multiple targets, rendering it a potential drug target for diseases such as heart failure.

Show MeSH
Related in: MedlinePlus