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MicroRNA-532-3p regulates mitochondrial fission through targeting apoptosis repressor with caspase recruitment domain in doxorubicin cardiotoxicity.

Wang JX, Zhang XJ, Feng C, Sun T, Wang K, Wang Y, Zhou LY, Li PF - Cell Death Dis (2015)

Bottom Line: DOX-induced mitochondrial fission required the activity of dynamin-related protein 1 (Drp1).In elucidating the molecular mechanism by which ARC was downregulated upon DOX treatment, miR-532-3p was found to directly target ARC and participated in DOX-induced mitochondrial fission and apoptosis.MiR-532-3p was not involved in DOX-induced apoptosis in cancer cells.

View Article: PubMed Central - PubMed

Affiliation: 1] National Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China [2] Institute for Translational Medicine, College of Medicine, Qingdao University, Qingdao, China.

ABSTRACT
Doxorubicin (DOX) is a wide-spectrum antitumor drug, but its clinical application is limited by its cardiotoxicity. However, the mechanisms underlying DOX-induced cardiomyopathy remain mostly unclear. Here we observed that apoptosis repressor with caspase recruitment domain (ARC) was downregulated in mouse heart and cardiomyocytes upon DOX treatment. Furthermore, enforced expression of ARC attenuated DOX-induced cardiomyocyte mitochondrial fission and apoptosis. ARC transgenic mice demonstrated reduced cardiotoxicity upon DOX administration. DOX-induced mitochondrial fission required the activity of dynamin-related protein 1 (Drp1). In elucidating the molecular mechanism by which ARC was downregulated upon DOX treatment, miR-532-3p was found to directly target ARC and participated in DOX-induced mitochondrial fission and apoptosis. MiR-532-3p was not involved in DOX-induced apoptosis in cancer cells. Taken together, these findings provide novel evidence that miR-532-3p and ARC constitute an antiapoptotic pathway that regulates DOX cardiotoxicity. Therefore, the development of new therapeutic strategies based on ARC and miR-532-3p is promising for overcoming the cardiotoxicity of chemotherapy for cancer therapy.

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MiR-532-3p participates in the regulation of ARC expression. (a) Analysis of ARC 3′UTR potential binding site for miR-532-3p by RNAhybrid. Potential complementary residues are shown in red. (b) The miR-532-3p levels in neonatal rat cardiomyocytes treated with 2 μM DOX at the indicated time. *P<0.05 versus control (untreated). (c) MiR-532-3p levels in mice administered with DOX or saline as described in Materials and Methods. n=5 mice per group. (d) ARC mRNA and protein levels in neonatal rat cardiomyocytes overexpressing miR-532-3p by transfecting with miR-532-3p mimics. (e) Kncokdown of endogenous miR-532-3p by transfecting its antagomirs attenuated decrease of ARC mRNA and protein levels upon DOX (2 μM) for 12 h in cardiomyocytes. (f) Luciferase activity detected in HEK-293 transfected with synthesized miR-532-3p mimic or mimic control, along with luciferase reporter constructs as indicated. (g) Luciferase activity of luciferase construct ARC 3′UTR-Wt is decreased upon DOX treatment in cardiomyocytes. Data are expressed as the mean±S.D., n=3 except in panel (c). *P<0.05
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fig4: MiR-532-3p participates in the regulation of ARC expression. (a) Analysis of ARC 3′UTR potential binding site for miR-532-3p by RNAhybrid. Potential complementary residues are shown in red. (b) The miR-532-3p levels in neonatal rat cardiomyocytes treated with 2 μM DOX at the indicated time. *P<0.05 versus control (untreated). (c) MiR-532-3p levels in mice administered with DOX or saline as described in Materials and Methods. n=5 mice per group. (d) ARC mRNA and protein levels in neonatal rat cardiomyocytes overexpressing miR-532-3p by transfecting with miR-532-3p mimics. (e) Kncokdown of endogenous miR-532-3p by transfecting its antagomirs attenuated decrease of ARC mRNA and protein levels upon DOX (2 μM) for 12 h in cardiomyocytes. (f) Luciferase activity detected in HEK-293 transfected with synthesized miR-532-3p mimic or mimic control, along with luciferase reporter constructs as indicated. (g) Luciferase activity of luciferase construct ARC 3′UTR-Wt is decreased upon DOX treatment in cardiomyocytes. Data are expressed as the mean±S.D., n=3 except in panel (c). *P<0.05

Mentions: MiRNA is able to suppress gene expression. To explore the underlying mechanism by which ARC is downregulated upon DOX, we tested whether miRNA can regulate ARC expression. We analyzed the 3′UTR of ARC using the RNAhybrid program and found some potential miRNAs. To explore which miRNA is involved in the regulation of ARC, we used quantitative real-time PCR (qRT-PCR) to detect miRNAs levels in response to DOX. Among several miRNAs, miR-532-3p was substantially increased (Supplementary Figure S4a). Accordingly, we focused on miR-532-3p. The potential miR-532-3p-binding site in ARC 3′UTR was shown in Figure 4a. The binding site was conserved among human, mouse and rat. The expression levels of miR-532-3p were increased in DOX-treated cardiomyocytes (Figure 4b) and in mice hearts (Figure 4c) exposed to DOX. First, we attempted to evaluate whether miR-532-3p modulated ARC expression. Enforced expression of miR-532-3p was confirmed by qRT-PCR (Supplementary Figure S4b). Overexpression of miR-532-3p led to an obvious reduction of ARC mRNA and protein levels in cardiomyocytes (Figure 4d). MiR-532-3p levels were reduced by its specific antagomir (Supplementary Figure S4c). In contrast, administration of the miR-532-3p antagomir could attenuate the decrease of ARC levels in response to DOX treatment (Figure 4e). Therefore, it seems that miR-532-3p modulates ARC expression by altering mRNA stability.


MicroRNA-532-3p regulates mitochondrial fission through targeting apoptosis repressor with caspase recruitment domain in doxorubicin cardiotoxicity.

Wang JX, Zhang XJ, Feng C, Sun T, Wang K, Wang Y, Zhou LY, Li PF - Cell Death Dis (2015)

MiR-532-3p participates in the regulation of ARC expression. (a) Analysis of ARC 3′UTR potential binding site for miR-532-3p by RNAhybrid. Potential complementary residues are shown in red. (b) The miR-532-3p levels in neonatal rat cardiomyocytes treated with 2 μM DOX at the indicated time. *P<0.05 versus control (untreated). (c) MiR-532-3p levels in mice administered with DOX or saline as described in Materials and Methods. n=5 mice per group. (d) ARC mRNA and protein levels in neonatal rat cardiomyocytes overexpressing miR-532-3p by transfecting with miR-532-3p mimics. (e) Kncokdown of endogenous miR-532-3p by transfecting its antagomirs attenuated decrease of ARC mRNA and protein levels upon DOX (2 μM) for 12 h in cardiomyocytes. (f) Luciferase activity detected in HEK-293 transfected with synthesized miR-532-3p mimic or mimic control, along with luciferase reporter constructs as indicated. (g) Luciferase activity of luciferase construct ARC 3′UTR-Wt is decreased upon DOX treatment in cardiomyocytes. Data are expressed as the mean±S.D., n=3 except in panel (c). *P<0.05
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4385919&req=5

fig4: MiR-532-3p participates in the regulation of ARC expression. (a) Analysis of ARC 3′UTR potential binding site for miR-532-3p by RNAhybrid. Potential complementary residues are shown in red. (b) The miR-532-3p levels in neonatal rat cardiomyocytes treated with 2 μM DOX at the indicated time. *P<0.05 versus control (untreated). (c) MiR-532-3p levels in mice administered with DOX or saline as described in Materials and Methods. n=5 mice per group. (d) ARC mRNA and protein levels in neonatal rat cardiomyocytes overexpressing miR-532-3p by transfecting with miR-532-3p mimics. (e) Kncokdown of endogenous miR-532-3p by transfecting its antagomirs attenuated decrease of ARC mRNA and protein levels upon DOX (2 μM) for 12 h in cardiomyocytes. (f) Luciferase activity detected in HEK-293 transfected with synthesized miR-532-3p mimic or mimic control, along with luciferase reporter constructs as indicated. (g) Luciferase activity of luciferase construct ARC 3′UTR-Wt is decreased upon DOX treatment in cardiomyocytes. Data are expressed as the mean±S.D., n=3 except in panel (c). *P<0.05
Mentions: MiRNA is able to suppress gene expression. To explore the underlying mechanism by which ARC is downregulated upon DOX, we tested whether miRNA can regulate ARC expression. We analyzed the 3′UTR of ARC using the RNAhybrid program and found some potential miRNAs. To explore which miRNA is involved in the regulation of ARC, we used quantitative real-time PCR (qRT-PCR) to detect miRNAs levels in response to DOX. Among several miRNAs, miR-532-3p was substantially increased (Supplementary Figure S4a). Accordingly, we focused on miR-532-3p. The potential miR-532-3p-binding site in ARC 3′UTR was shown in Figure 4a. The binding site was conserved among human, mouse and rat. The expression levels of miR-532-3p were increased in DOX-treated cardiomyocytes (Figure 4b) and in mice hearts (Figure 4c) exposed to DOX. First, we attempted to evaluate whether miR-532-3p modulated ARC expression. Enforced expression of miR-532-3p was confirmed by qRT-PCR (Supplementary Figure S4b). Overexpression of miR-532-3p led to an obvious reduction of ARC mRNA and protein levels in cardiomyocytes (Figure 4d). MiR-532-3p levels were reduced by its specific antagomir (Supplementary Figure S4c). In contrast, administration of the miR-532-3p antagomir could attenuate the decrease of ARC levels in response to DOX treatment (Figure 4e). Therefore, it seems that miR-532-3p modulates ARC expression by altering mRNA stability.

Bottom Line: DOX-induced mitochondrial fission required the activity of dynamin-related protein 1 (Drp1).In elucidating the molecular mechanism by which ARC was downregulated upon DOX treatment, miR-532-3p was found to directly target ARC and participated in DOX-induced mitochondrial fission and apoptosis.MiR-532-3p was not involved in DOX-induced apoptosis in cancer cells.

View Article: PubMed Central - PubMed

Affiliation: 1] National Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China [2] Institute for Translational Medicine, College of Medicine, Qingdao University, Qingdao, China.

ABSTRACT
Doxorubicin (DOX) is a wide-spectrum antitumor drug, but its clinical application is limited by its cardiotoxicity. However, the mechanisms underlying DOX-induced cardiomyopathy remain mostly unclear. Here we observed that apoptosis repressor with caspase recruitment domain (ARC) was downregulated in mouse heart and cardiomyocytes upon DOX treatment. Furthermore, enforced expression of ARC attenuated DOX-induced cardiomyocyte mitochondrial fission and apoptosis. ARC transgenic mice demonstrated reduced cardiotoxicity upon DOX administration. DOX-induced mitochondrial fission required the activity of dynamin-related protein 1 (Drp1). In elucidating the molecular mechanism by which ARC was downregulated upon DOX treatment, miR-532-3p was found to directly target ARC and participated in DOX-induced mitochondrial fission and apoptosis. MiR-532-3p was not involved in DOX-induced apoptosis in cancer cells. Taken together, these findings provide novel evidence that miR-532-3p and ARC constitute an antiapoptotic pathway that regulates DOX cardiotoxicity. Therefore, the development of new therapeutic strategies based on ARC and miR-532-3p is promising for overcoming the cardiotoxicity of chemotherapy for cancer therapy.

Show MeSH
Related in: MedlinePlus