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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 directly targets 3′-UTR of Camk2d, Ncx1, and Nfatc3.(A) Schematic diagram of the pmirGLO chimeric vector indicating where the exact complement target sequences for miR-185 or mutant sequences were cloned into the 3′-UTR of the luciferase gene. (B-D) The relative positions of predicted binding sites for mouse miR-185 in the 3′-UTR of the target mRNAs (upper) and quantitative analysis of luciferase activity of the reporter constructs (bottom). HEK-293 cells were transfected with miR-185 in addition to the reporter constructs with WT or mutated 3′-UTR. After 72 h following transfection, cells were lysed for dual luciferase assay. Hsa, human; Mmu, mouse; Rno, rat; Ptr, chimpanzee. Data represent the mean ± SEM; **P < 0.001, or NS (not significant), N = 3.
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pone.0122509.g003: miR-185 directly targets 3′-UTR of Camk2d, Ncx1, and Nfatc3.(A) Schematic diagram of the pmirGLO chimeric vector indicating where the exact complement target sequences for miR-185 or mutant sequences were cloned into the 3′-UTR of the luciferase gene. (B-D) The relative positions of predicted binding sites for mouse miR-185 in the 3′-UTR of the target mRNAs (upper) and quantitative analysis of luciferase activity of the reporter constructs (bottom). HEK-293 cells were transfected with miR-185 in addition to the reporter constructs with WT or mutated 3′-UTR. After 72 h following transfection, cells were lysed for dual luciferase assay. Hsa, human; Mmu, mouse; Rno, rat; Ptr, chimpanzee. Data represent the mean ± SEM; **P < 0.001, or NS (not significant), N = 3.

Mentions: Next, we determined whether Camk2d, Ncx1, and Nfatc3 are the direct targets of miR-185. While Nfatc3 harbors a single binding site for miR-185 in the 3′-UTR (Fig. 3C), there are multiple putative binding sites in the 3′-UTRs of Camk2d and Ncx1 in mouse (Fig. 3B and 3D). To critically examine the interactions between miR-185 and the putative targets, we measured the activity of the luciferase reporter when linked to either the WT or mutant 3′-UTR of each target (Fig. 3A). The results showed that luciferase activity at Site 2 of Camk2d, and at Site 1 of Nfatc3 and Ncx1, which are highly conserved across species, was significantly suppressed by miR-185. In the same context, mutations of the target sites in the 3′-UTR completely blocked the inhibitory effects of miR-185, suggesting that the identified sites, shown in Figs. 3B-3D, are the direct target sites for miR-185 regulation. Other predicted targets, including Ctf1, Elk1, and Mtpn, were false positives (Figure H in S1 File). Collectively, our results suggest that miR-185 have multiple targets in the Ca2+-dependent cardiac hypertrophy signaling pathway.


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 directly targets 3′-UTR of Camk2d, Ncx1, and Nfatc3.(A) Schematic diagram of the pmirGLO chimeric vector indicating where the exact complement target sequences for miR-185 or mutant sequences were cloned into the 3′-UTR of the luciferase gene. (B-D) The relative positions of predicted binding sites for mouse miR-185 in the 3′-UTR of the target mRNAs (upper) and quantitative analysis of luciferase activity of the reporter constructs (bottom). HEK-293 cells were transfected with miR-185 in addition to the reporter constructs with WT or mutated 3′-UTR. After 72 h following transfection, cells were lysed for dual luciferase assay. Hsa, human; Mmu, mouse; Rno, rat; Ptr, chimpanzee. Data represent the mean ± SEM; **P < 0.001, or NS (not significant), N = 3.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0122509.g003: miR-185 directly targets 3′-UTR of Camk2d, Ncx1, and Nfatc3.(A) Schematic diagram of the pmirGLO chimeric vector indicating where the exact complement target sequences for miR-185 or mutant sequences were cloned into the 3′-UTR of the luciferase gene. (B-D) The relative positions of predicted binding sites for mouse miR-185 in the 3′-UTR of the target mRNAs (upper) and quantitative analysis of luciferase activity of the reporter constructs (bottom). HEK-293 cells were transfected with miR-185 in addition to the reporter constructs with WT or mutated 3′-UTR. After 72 h following transfection, cells were lysed for dual luciferase assay. Hsa, human; Mmu, mouse; Rno, rat; Ptr, chimpanzee. Data represent the mean ± SEM; **P < 0.001, or NS (not significant), N = 3.
Mentions: Next, we determined whether Camk2d, Ncx1, and Nfatc3 are the direct targets of miR-185. While Nfatc3 harbors a single binding site for miR-185 in the 3′-UTR (Fig. 3C), there are multiple putative binding sites in the 3′-UTRs of Camk2d and Ncx1 in mouse (Fig. 3B and 3D). To critically examine the interactions between miR-185 and the putative targets, we measured the activity of the luciferase reporter when linked to either the WT or mutant 3′-UTR of each target (Fig. 3A). The results showed that luciferase activity at Site 2 of Camk2d, and at Site 1 of Nfatc3 and Ncx1, which are highly conserved across species, was significantly suppressed by miR-185. In the same context, mutations of the target sites in the 3′-UTR completely blocked the inhibitory effects of miR-185, suggesting that the identified sites, shown in Figs. 3B-3D, are the direct target sites for miR-185 regulation. Other predicted targets, including Ctf1, Elk1, and Mtpn, were false positives (Figure H in S1 File). Collectively, our results suggest that miR-185 have multiple targets in the Ca2+-dependent cardiac hypertrophy signaling pathway.

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