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MicroRNA-30b-mediated regulation of catalase expression in human ARPE-19 cells.

Haque R, Chun E, Howell JC, Sengupta T, Chen D, Kim H - PLoS ONE (2012)

Bottom Line: Here, we demonstrated that a sublethal dose of H(2)O(2) (200 µM) up-regulated the expression of miR-30b, a member of the miR-30 family, which inhibited the expression of endogenous catalase both at the transcript and protein levels.However, antisense (antagomirs) of miR-30b was not only found to suppress the miR-30b mimics-mediated inhibitions, but also to dramatically increase the expression of catalase even under an oxidant environment.We propose that a microRNA antisense approach could enhance cytoprotective mechanisms against oxidative stress by increasing the antioxidant defense system.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Emory University School of Medicine, Atlanta, Georgia, United States of America. rhaque@emory.edu

ABSTRACT

Background: Oxidative injury to retinal pigment epithelium (RPE) and retinal photoreceptors has been linked to a number of retinal diseases, including age-related macular degeneration (AMD). Reactive oxygen species (ROS)-mediated gene expression has been extensively studied at transcriptional levels. Also, the post-transcriptional control of gene expression at the level of translational regulation has been recently reported. However, the microRNA (miRNA/miR)-mediated post-transcriptional regulation in human RPE cells has not been thoroughly looked at. Increasing evidence points to a potential role of miRNAs in diverse physiological processes.

Methodology/principal findings: We demonstrated for the first time in a human retinal pigment epithelial cell line (ARPE-19) that the post-transcriptional control of gene expression via miRNA modulation regulates human catalase, an important and potent component of cell's antioxidant defensive network, which detoxifies hydrogen peroxide (H(2)O(2)) radicals. Exposure to several stress-inducing agents including H(2)O(2) has been reported to alter miRNA expression profile. Here, we demonstrated that a sublethal dose of H(2)O(2) (200 µM) up-regulated the expression of miR-30b, a member of the miR-30 family, which inhibited the expression of endogenous catalase both at the transcript and protein levels. However, antisense (antagomirs) of miR-30b was not only found to suppress the miR-30b mimics-mediated inhibitions, but also to dramatically increase the expression of catalase even under an oxidant environment.

Conclusions/significance: We propose that a microRNA antisense approach could enhance cytoprotective mechanisms against oxidative stress by increasing the antioxidant defense system.

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Related in: MedlinePlus

Antagomirs of miR-30b protect ARPE-19 cells from miR-30b mimics-mediated inhibition of catalase expression under oxidative condition.Cells were transfected with NC (20 nM), mimics (20 nM), antagomirs (50 nM), or mimics+antagomirs, and incubated for 24 h before harvesting for total RNA extraction; H2O2 (200 uM) was added for the last 18 h. Catalase mRNA levels were expressed relative to Hprt mRNA with the value of the scrambled miRNA-transfected control (NC) set to 1. Values are presented as mean ± SEM; n = 4. *p<0.05 vs. control (NC), **p<0.001 vs. NC/mimics/mimics+H2O2/mimics+antagomirs+H2O2, §p<0.001 vs. mimics/mimics+H2O2, # p<0.05 vs. antagomirs, ## p<0.001 vs. H2O2/NC+ H2O2.
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pone-0042542-g007: Antagomirs of miR-30b protect ARPE-19 cells from miR-30b mimics-mediated inhibition of catalase expression under oxidative condition.Cells were transfected with NC (20 nM), mimics (20 nM), antagomirs (50 nM), or mimics+antagomirs, and incubated for 24 h before harvesting for total RNA extraction; H2O2 (200 uM) was added for the last 18 h. Catalase mRNA levels were expressed relative to Hprt mRNA with the value of the scrambled miRNA-transfected control (NC) set to 1. Values are presented as mean ± SEM; n = 4. *p<0.05 vs. control (NC), **p<0.001 vs. NC/mimics/mimics+H2O2/mimics+antagomirs+H2O2, §p<0.001 vs. mimics/mimics+H2O2, # p<0.05 vs. antagomirs, ## p<0.001 vs. H2O2/NC+ H2O2.

Mentions: To determine the protective effect of antagomirs on catalase expression under oxidative stress, ARPE-19 cells were transfected with scrambled miRNA (NC, 20 nM), mimics (20 nM), antagomirs (50 nM), or mimics and antagomirs together, and then the cells were exposed to oxidative stress (Figure 7). H2O2 in presence or absence of NC significantly increased the level of catalase mRNA expression (p<0.05) as compared with the control (NC). miR-30b mimics not only significantly inhibited catalase expression (p<0.05) when compared with the NC, but also very significantly (p<0.001) suppressed the H2O2-stimulated expression of catalase. However, what is interesting is that miR-30b antagomirs not only protected miR-30b mimics-mediated inhibition of catalase expression (p<0.001), but also significantly (p<0.001) enhanced its expression even when cells were stressed with H2O2. Furthermore, mir-30b antagomirs-mediated increase of catalase expression was significantly higher than the cells treated with H2O2 (p = 0.009). However, co-transfection of miR-30b antagomirs together with the mimics of miR-30b in presence or absence of H2O2 resulted in significant increase of catalase expression as compared with the NC (p<0.05) or with the groups mimics/mimics+H2O2 (p<0.001).


MicroRNA-30b-mediated regulation of catalase expression in human ARPE-19 cells.

Haque R, Chun E, Howell JC, Sengupta T, Chen D, Kim H - PLoS ONE (2012)

Antagomirs of miR-30b protect ARPE-19 cells from miR-30b mimics-mediated inhibition of catalase expression under oxidative condition.Cells were transfected with NC (20 nM), mimics (20 nM), antagomirs (50 nM), or mimics+antagomirs, and incubated for 24 h before harvesting for total RNA extraction; H2O2 (200 uM) was added for the last 18 h. Catalase mRNA levels were expressed relative to Hprt mRNA with the value of the scrambled miRNA-transfected control (NC) set to 1. Values are presented as mean ± SEM; n = 4. *p<0.05 vs. control (NC), **p<0.001 vs. NC/mimics/mimics+H2O2/mimics+antagomirs+H2O2, §p<0.001 vs. mimics/mimics+H2O2, # p<0.05 vs. antagomirs, ## p<0.001 vs. H2O2/NC+ H2O2.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3412823&req=5

pone-0042542-g007: Antagomirs of miR-30b protect ARPE-19 cells from miR-30b mimics-mediated inhibition of catalase expression under oxidative condition.Cells were transfected with NC (20 nM), mimics (20 nM), antagomirs (50 nM), or mimics+antagomirs, and incubated for 24 h before harvesting for total RNA extraction; H2O2 (200 uM) was added for the last 18 h. Catalase mRNA levels were expressed relative to Hprt mRNA with the value of the scrambled miRNA-transfected control (NC) set to 1. Values are presented as mean ± SEM; n = 4. *p<0.05 vs. control (NC), **p<0.001 vs. NC/mimics/mimics+H2O2/mimics+antagomirs+H2O2, §p<0.001 vs. mimics/mimics+H2O2, # p<0.05 vs. antagomirs, ## p<0.001 vs. H2O2/NC+ H2O2.
Mentions: To determine the protective effect of antagomirs on catalase expression under oxidative stress, ARPE-19 cells were transfected with scrambled miRNA (NC, 20 nM), mimics (20 nM), antagomirs (50 nM), or mimics and antagomirs together, and then the cells were exposed to oxidative stress (Figure 7). H2O2 in presence or absence of NC significantly increased the level of catalase mRNA expression (p<0.05) as compared with the control (NC). miR-30b mimics not only significantly inhibited catalase expression (p<0.05) when compared with the NC, but also very significantly (p<0.001) suppressed the H2O2-stimulated expression of catalase. However, what is interesting is that miR-30b antagomirs not only protected miR-30b mimics-mediated inhibition of catalase expression (p<0.001), but also significantly (p<0.001) enhanced its expression even when cells were stressed with H2O2. Furthermore, mir-30b antagomirs-mediated increase of catalase expression was significantly higher than the cells treated with H2O2 (p = 0.009). However, co-transfection of miR-30b antagomirs together with the mimics of miR-30b in presence or absence of H2O2 resulted in significant increase of catalase expression as compared with the NC (p<0.05) or with the groups mimics/mimics+H2O2 (p<0.001).

Bottom Line: Here, we demonstrated that a sublethal dose of H(2)O(2) (200 µM) up-regulated the expression of miR-30b, a member of the miR-30 family, which inhibited the expression of endogenous catalase both at the transcript and protein levels.However, antisense (antagomirs) of miR-30b was not only found to suppress the miR-30b mimics-mediated inhibitions, but also to dramatically increase the expression of catalase even under an oxidant environment.We propose that a microRNA antisense approach could enhance cytoprotective mechanisms against oxidative stress by increasing the antioxidant defense system.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Emory University School of Medicine, Atlanta, Georgia, United States of America. rhaque@emory.edu

ABSTRACT

Background: Oxidative injury to retinal pigment epithelium (RPE) and retinal photoreceptors has been linked to a number of retinal diseases, including age-related macular degeneration (AMD). Reactive oxygen species (ROS)-mediated gene expression has been extensively studied at transcriptional levels. Also, the post-transcriptional control of gene expression at the level of translational regulation has been recently reported. However, the microRNA (miRNA/miR)-mediated post-transcriptional regulation in human RPE cells has not been thoroughly looked at. Increasing evidence points to a potential role of miRNAs in diverse physiological processes.

Methodology/principal findings: We demonstrated for the first time in a human retinal pigment epithelial cell line (ARPE-19) that the post-transcriptional control of gene expression via miRNA modulation regulates human catalase, an important and potent component of cell's antioxidant defensive network, which detoxifies hydrogen peroxide (H(2)O(2)) radicals. Exposure to several stress-inducing agents including H(2)O(2) has been reported to alter miRNA expression profile. Here, we demonstrated that a sublethal dose of H(2)O(2) (200 µM) up-regulated the expression of miR-30b, a member of the miR-30 family, which inhibited the expression of endogenous catalase both at the transcript and protein levels. However, antisense (antagomirs) of miR-30b was not only found to suppress the miR-30b mimics-mediated inhibitions, but also to dramatically increase the expression of catalase even under an oxidant environment.

Conclusions/significance: We propose that a microRNA antisense approach could enhance cytoprotective mechanisms against oxidative stress by increasing the antioxidant defense system.

Show MeSH
Related in: MedlinePlus