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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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Related in: MedlinePlus

miR-185 negatively regulates the activity of CaMKIIδ in NRVMs.(A and B) 72 h after transfection of miR-185 mimic or miR-185 inhibitor, the level of p-CaMKIIδ and total CaMKIIδ were analyzed by western blotting. GAPDH was used as a loading control. (C) Western blotting showing p-PLB (Thr-17) and PLB protein expression in NRVMs transfected with NC or miR-185. 24 h after transfection, NRVMs were stimulated with ET-1 (10 nmol/L) for 48 h. The blots were stripped for 30 min and reprobed with PLB for loading control. The data are expressed as mean ± SEM; *P < 0.05, **P < 0.001, N = 3.
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pone.0122509.g005: miR-185 negatively regulates the activity of CaMKIIδ in NRVMs.(A and B) 72 h after transfection of miR-185 mimic or miR-185 inhibitor, the level of p-CaMKIIδ and total CaMKIIδ were analyzed by western blotting. GAPDH was used as a loading control. (C) Western blotting showing p-PLB (Thr-17) and PLB protein expression in NRVMs transfected with NC or miR-185. 24 h after transfection, NRVMs were stimulated with ET-1 (10 nmol/L) for 48 h. The blots were stripped for 30 min and reprobed with PLB for loading control. The data are expressed as mean ± SEM; *P < 0.05, **P < 0.001, N = 3.

Mentions: Binding of Ca2+/calmodulin to the regulatory domain leads activates CaMKIIδ, and the activated enzyme is subsequently autophosphorylated at Thr-286/287, rendering the kinase constitutively active [27]. Based on our identification of Camk2d as a target of miR-185 (Fig. 3B), we performed western blotting to examine the level of p-CaMKIIδ after transfection of miR-185 mimic or miR-185 inhibitor. As expected, the levels of CaMKIIδ phosphorylation at Thr-286 and total CaMKIIδ were significantly lower in miR-185-overexpressing cardiomyocytes compared with the levels in the control group (Fig. 5A), while inhibition of miR-185 significantly up-regulated phosphorylation of CaMKIIδ at Thr-286 and the amount of total CaMKIIδ (Fig. 5B).


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 the activity of CaMKIIδ in NRVMs.(A and B) 72 h after transfection of miR-185 mimic or miR-185 inhibitor, the level of p-CaMKIIδ and total CaMKIIδ were analyzed by western blotting. GAPDH was used as a loading control. (C) Western blotting showing p-PLB (Thr-17) and PLB protein expression in NRVMs transfected with NC or miR-185. 24 h after transfection, NRVMs were stimulated with ET-1 (10 nmol/L) for 48 h. The blots were stripped for 30 min and reprobed with PLB for loading control. The data are expressed as mean ± SEM; *P < 0.05, **P < 0.001, N = 3.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0122509.g005: miR-185 negatively regulates the activity of CaMKIIδ in NRVMs.(A and B) 72 h after transfection of miR-185 mimic or miR-185 inhibitor, the level of p-CaMKIIδ and total CaMKIIδ were analyzed by western blotting. GAPDH was used as a loading control. (C) Western blotting showing p-PLB (Thr-17) and PLB protein expression in NRVMs transfected with NC or miR-185. 24 h after transfection, NRVMs were stimulated with ET-1 (10 nmol/L) for 48 h. The blots were stripped for 30 min and reprobed with PLB for loading control. The data are expressed as mean ± SEM; *P < 0.05, **P < 0.001, N = 3.
Mentions: Binding of Ca2+/calmodulin to the regulatory domain leads activates CaMKIIδ, and the activated enzyme is subsequently autophosphorylated at Thr-286/287, rendering the kinase constitutively active [27]. Based on our identification of Camk2d as a target of miR-185 (Fig. 3B), we performed western blotting to examine the level of p-CaMKIIδ after transfection of miR-185 mimic or miR-185 inhibitor. As expected, the levels of CaMKIIδ phosphorylation at Thr-286 and total CaMKIIδ were significantly lower in miR-185-overexpressing cardiomyocytes compared with the levels in the control group (Fig. 5A), while inhibition of miR-185 significantly up-regulated phosphorylation of CaMKIIδ at Thr-286 and the amount of total CaMKIIδ (Fig. 5B).

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