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Ischemic preconditioning potentiates the protective effect of stem cells through secretion of exosomes by targeting Mecp2 via miR-22.

Feng Y, Huang W, Wani M, Yu X, Ashraf M - PLoS ONE (2014)

Bottom Line: Furthermore, the anti-apoptotic effect of miR-22 was mediated by direct targeting of methyl CpG binding protein 2 (Mecp2).In vivo data showed that delivery of Exo(IPC) significantly reduced cardiac fibrosis.Our data identified a significant benefit of Exo(IPC) for the treatment of cardiac diseases by targeting Mecp2 via miR-22.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Center of Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangdong Provincial Cardiovascular Institute, Southern Medical University, Guangzhou, China ; Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, Ohio, United States of America.

ABSTRACT
Mesenchymal stem cells (MSCs) have potential application for the treatment of ischemic heart diseases. Besides differentiation properties, MSCs protect ischemic cardiomyocytes by secretion of paracrine factors. In this study, we found exosomes enriched with miR-22 were secreted by MSCs following ischemic preconditioning (Exo(IPC)) and mobilized to cardiomyocytes where they reduced their apoptosis due to ischemia. Interestingly, by time-lapse imaging, we for the first time captured the dynamic shedding of miR-22 loaded exosomes from cytosol to extracellular space. Furthermore, the anti-apoptotic effect of miR-22 was mediated by direct targeting of methyl CpG binding protein 2 (Mecp2). In vivo data showed that delivery of Exo(IPC) significantly reduced cardiac fibrosis. Our data identified a significant benefit of Exo(IPC) for the treatment of cardiac diseases by targeting Mecp2 via miR-22.

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miR-22 targets Mecp2.A. Target scan showed that Mecp2 is predicted target of miR-22 with 4 potential binding sites on its 3′UTR. B. Western blot was performed in MSCs post transfection of microRNA scramble (miR-Scr), miR-22 mimic (100 nM) and miR-22 LNA inhibitor (50 nM) in MSCs. C. Luciferase activity was employed in MSCs post transfection of microRNA scramble (miR-Scr), miR-22 mimic (100 nM) and miR-22 LNA inhibitor (50 nM). (*) denotes P<0.05 vs. control for significant difference (n = 3). (#) denotes P<0.05 vs. miR-22 mimic for significant difference (n = 3). D. Western blot showing upregulation of Mecp2 in infarcted hearts. E. Western blot showing successful knockdown of Mecp2 in infarcted hearts with siRNA (si-Mecp2) F. TUNEL assay showing reduction of apoptosis in ischemic cardiomyocytes by si-Mecp2. (*) denotes P<0.05 vs. control for significant difference (n = 3 for each group). (#) denotes P<0.05 vs. miR-22 mimic for significant difference (n = 3). G. Western blot showing downregulation of Mecp2 in infarcted hearts by miR-22 mimic. H. TUNEL assay showing reduction of apoptosis in ischemic cardiomyocytes by miR-22 mimic. (*) denotes P<0.05 vs. control for significant difference. (#) denotes P<0.05 vs. miR-22 mimic for significant difference (n = 3 for each group).
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pone-0088685-g004: miR-22 targets Mecp2.A. Target scan showed that Mecp2 is predicted target of miR-22 with 4 potential binding sites on its 3′UTR. B. Western blot was performed in MSCs post transfection of microRNA scramble (miR-Scr), miR-22 mimic (100 nM) and miR-22 LNA inhibitor (50 nM) in MSCs. C. Luciferase activity was employed in MSCs post transfection of microRNA scramble (miR-Scr), miR-22 mimic (100 nM) and miR-22 LNA inhibitor (50 nM). (*) denotes P<0.05 vs. control for significant difference (n = 3). (#) denotes P<0.05 vs. miR-22 mimic for significant difference (n = 3). D. Western blot showing upregulation of Mecp2 in infarcted hearts. E. Western blot showing successful knockdown of Mecp2 in infarcted hearts with siRNA (si-Mecp2) F. TUNEL assay showing reduction of apoptosis in ischemic cardiomyocytes by si-Mecp2. (*) denotes P<0.05 vs. control for significant difference (n = 3 for each group). (#) denotes P<0.05 vs. miR-22 mimic for significant difference (n = 3). G. Western blot showing downregulation of Mecp2 in infarcted hearts by miR-22 mimic. H. TUNEL assay showing reduction of apoptosis in ischemic cardiomyocytes by miR-22 mimic. (*) denotes P<0.05 vs. control for significant difference. (#) denotes P<0.05 vs. miR-22 mimic for significant difference (n = 3 for each group).

Mentions: In order to investigate the direct downstream effectors/targets by which miR-22 exerts its function, we used computational algorithms to predict potential miR-24 target genes. Computational analysis showed Mecp2 is a potential target of miR-22 by binding on the 3′UTR (Fig. 4A). Therefore, we interrogated the relationship of miR-22 and Mecp2 in cardiomyocytes. As shown in (Fig. 4B), transfection of miR-22 mimic downregulated Mecp2 in cardiomyocytes while transfection of miR-22 inhibitor upregulated Mecp2 in cardiomyocytes. Moreover, luciferase reporter assay in which the luciferase reporter gene was fused to the wild-type Mecp2 3′UTR showed that luciferase activity in MSCs transfected with miR-22 mimic was downregulated while it was upregulated in MSCs transfected with miR-22 inhibitor (Fig. 4C). From these data, we concluded that the miR-22 targets Mecp2. Moreover, we found Mecp2 was upregulated in infarcted hearts as shown in (Fig. 4D). Furthermore, knockdown of Mecp2 by specific siRNAs in infarcted hearts (Fig. 4E) reduced apoptosis in the ischemic myocardium (Fig. 4F). Similarly, delivery of miR-22 mimic significantly downregulated Mecp2 (Fig. 4G) and ameliorated apoptosis in ischemic myocardium (Fig. 4H). Taken together, these data showed Mecp2 is a functional target of miR-22.


Ischemic preconditioning potentiates the protective effect of stem cells through secretion of exosomes by targeting Mecp2 via miR-22.

Feng Y, Huang W, Wani M, Yu X, Ashraf M - PLoS ONE (2014)

miR-22 targets Mecp2.A. Target scan showed that Mecp2 is predicted target of miR-22 with 4 potential binding sites on its 3′UTR. B. Western blot was performed in MSCs post transfection of microRNA scramble (miR-Scr), miR-22 mimic (100 nM) and miR-22 LNA inhibitor (50 nM) in MSCs. C. Luciferase activity was employed in MSCs post transfection of microRNA scramble (miR-Scr), miR-22 mimic (100 nM) and miR-22 LNA inhibitor (50 nM). (*) denotes P<0.05 vs. control for significant difference (n = 3). (#) denotes P<0.05 vs. miR-22 mimic for significant difference (n = 3). D. Western blot showing upregulation of Mecp2 in infarcted hearts. E. Western blot showing successful knockdown of Mecp2 in infarcted hearts with siRNA (si-Mecp2) F. TUNEL assay showing reduction of apoptosis in ischemic cardiomyocytes by si-Mecp2. (*) denotes P<0.05 vs. control for significant difference (n = 3 for each group). (#) denotes P<0.05 vs. miR-22 mimic for significant difference (n = 3). G. Western blot showing downregulation of Mecp2 in infarcted hearts by miR-22 mimic. H. TUNEL assay showing reduction of apoptosis in ischemic cardiomyocytes by miR-22 mimic. (*) denotes P<0.05 vs. control for significant difference. (#) denotes P<0.05 vs. miR-22 mimic for significant difference (n = 3 for each group).
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pone-0088685-g004: miR-22 targets Mecp2.A. Target scan showed that Mecp2 is predicted target of miR-22 with 4 potential binding sites on its 3′UTR. B. Western blot was performed in MSCs post transfection of microRNA scramble (miR-Scr), miR-22 mimic (100 nM) and miR-22 LNA inhibitor (50 nM) in MSCs. C. Luciferase activity was employed in MSCs post transfection of microRNA scramble (miR-Scr), miR-22 mimic (100 nM) and miR-22 LNA inhibitor (50 nM). (*) denotes P<0.05 vs. control for significant difference (n = 3). (#) denotes P<0.05 vs. miR-22 mimic for significant difference (n = 3). D. Western blot showing upregulation of Mecp2 in infarcted hearts. E. Western blot showing successful knockdown of Mecp2 in infarcted hearts with siRNA (si-Mecp2) F. TUNEL assay showing reduction of apoptosis in ischemic cardiomyocytes by si-Mecp2. (*) denotes P<0.05 vs. control for significant difference (n = 3 for each group). (#) denotes P<0.05 vs. miR-22 mimic for significant difference (n = 3). G. Western blot showing downregulation of Mecp2 in infarcted hearts by miR-22 mimic. H. TUNEL assay showing reduction of apoptosis in ischemic cardiomyocytes by miR-22 mimic. (*) denotes P<0.05 vs. control for significant difference. (#) denotes P<0.05 vs. miR-22 mimic for significant difference (n = 3 for each group).
Mentions: In order to investigate the direct downstream effectors/targets by which miR-22 exerts its function, we used computational algorithms to predict potential miR-24 target genes. Computational analysis showed Mecp2 is a potential target of miR-22 by binding on the 3′UTR (Fig. 4A). Therefore, we interrogated the relationship of miR-22 and Mecp2 in cardiomyocytes. As shown in (Fig. 4B), transfection of miR-22 mimic downregulated Mecp2 in cardiomyocytes while transfection of miR-22 inhibitor upregulated Mecp2 in cardiomyocytes. Moreover, luciferase reporter assay in which the luciferase reporter gene was fused to the wild-type Mecp2 3′UTR showed that luciferase activity in MSCs transfected with miR-22 mimic was downregulated while it was upregulated in MSCs transfected with miR-22 inhibitor (Fig. 4C). From these data, we concluded that the miR-22 targets Mecp2. Moreover, we found Mecp2 was upregulated in infarcted hearts as shown in (Fig. 4D). Furthermore, knockdown of Mecp2 by specific siRNAs in infarcted hearts (Fig. 4E) reduced apoptosis in the ischemic myocardium (Fig. 4F). Similarly, delivery of miR-22 mimic significantly downregulated Mecp2 (Fig. 4G) and ameliorated apoptosis in ischemic myocardium (Fig. 4H). Taken together, these data showed Mecp2 is a functional target of miR-22.

Bottom Line: Furthermore, the anti-apoptotic effect of miR-22 was mediated by direct targeting of methyl CpG binding protein 2 (Mecp2).In vivo data showed that delivery of Exo(IPC) significantly reduced cardiac fibrosis.Our data identified a significant benefit of Exo(IPC) for the treatment of cardiac diseases by targeting Mecp2 via miR-22.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Center of Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangdong Provincial Cardiovascular Institute, Southern Medical University, Guangzhou, China ; Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, Ohio, United States of America.

ABSTRACT
Mesenchymal stem cells (MSCs) have potential application for the treatment of ischemic heart diseases. Besides differentiation properties, MSCs protect ischemic cardiomyocytes by secretion of paracrine factors. In this study, we found exosomes enriched with miR-22 were secreted by MSCs following ischemic preconditioning (Exo(IPC)) and mobilized to cardiomyocytes where they reduced their apoptosis due to ischemia. Interestingly, by time-lapse imaging, we for the first time captured the dynamic shedding of miR-22 loaded exosomes from cytosol to extracellular space. Furthermore, the anti-apoptotic effect of miR-22 was mediated by direct targeting of methyl CpG binding protein 2 (Mecp2). In vivo data showed that delivery of Exo(IPC) significantly reduced cardiac fibrosis. Our data identified a significant benefit of Exo(IPC) for the treatment of cardiac diseases by targeting Mecp2 via miR-22.

Show MeSH
Related in: MedlinePlus