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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

Effect of mimics and antagomirs on cell viability assessed by CellTiter-Blue in ARPE-19.ARPE-19 cells were transfected with mimics (20 nM) and antagomirs (50 nM) in presence or absence of H2O2 treatment, and incubated for 24 h before adding the CellTiter-Blue reagents; H2O2 (200 µM) was added for the last 18 h. NC (20 nM) and H2O2 (200 µM) alone were also used as controls. Data were analyzed using analysis of variance (ANOVA) with Student–Newman–Keuls multiple comparison tests and are expressed as a percentage of the untreated control. The CellTiter-Blue values as labeled by an asterisk are significantly different from the control (p<0.05). Values are presented as mean ± SEM; n = 5 per group.
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pone-0042542-g003: Effect of mimics and antagomirs on cell viability assessed by CellTiter-Blue in ARPE-19.ARPE-19 cells were transfected with mimics (20 nM) and antagomirs (50 nM) in presence or absence of H2O2 treatment, and incubated for 24 h before adding the CellTiter-Blue reagents; H2O2 (200 µM) was added for the last 18 h. NC (20 nM) and H2O2 (200 µM) alone were also used as controls. Data were analyzed using analysis of variance (ANOVA) with Student–Newman–Keuls multiple comparison tests and are expressed as a percentage of the untreated control. The CellTiter-Blue values as labeled by an asterisk are significantly different from the control (p<0.05). Values are presented as mean ± SEM; n = 5 per group.

Mentions: To eliminate the possibility that the inhibitory action of miRNA mimics on catalase expression in presence of H2O2 was due to the toxic effect of H2O2, we measured the cellular viability at 24 h and found no significant changes in the cells transfected with either mimics (20 nM) or antagomirs (50 nM) of miR-30b, when compared to negative control (NC). H2O2 alone and together with miR-30b mimics significantly (p<0.05) reduced the cell viability, as compared to NC. H2O2 alone also showed a significant difference in cell viability when compared with mimics (p = 0.008) and antagomirs (p<0.002). However, miR-30b antagomirs significantly rescued H2O2-mediated cell death, as compared to cells treated with H2O2 alone (p = 0.004) or H2O2 with the mimics (p = 0.002) [Figure 3].


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)

Effect of mimics and antagomirs on cell viability assessed by CellTiter-Blue in ARPE-19.ARPE-19 cells were transfected with mimics (20 nM) and antagomirs (50 nM) in presence or absence of H2O2 treatment, and incubated for 24 h before adding the CellTiter-Blue reagents; H2O2 (200 µM) was added for the last 18 h. NC (20 nM) and H2O2 (200 µM) alone were also used as controls. Data were analyzed using analysis of variance (ANOVA) with Student–Newman–Keuls multiple comparison tests and are expressed as a percentage of the untreated control. The CellTiter-Blue values as labeled by an asterisk are significantly different from the control (p<0.05). Values are presented as mean ± SEM; n = 5 per group.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3412823&req=5

pone-0042542-g003: Effect of mimics and antagomirs on cell viability assessed by CellTiter-Blue in ARPE-19.ARPE-19 cells were transfected with mimics (20 nM) and antagomirs (50 nM) in presence or absence of H2O2 treatment, and incubated for 24 h before adding the CellTiter-Blue reagents; H2O2 (200 µM) was added for the last 18 h. NC (20 nM) and H2O2 (200 µM) alone were also used as controls. Data were analyzed using analysis of variance (ANOVA) with Student–Newman–Keuls multiple comparison tests and are expressed as a percentage of the untreated control. The CellTiter-Blue values as labeled by an asterisk are significantly different from the control (p<0.05). Values are presented as mean ± SEM; n = 5 per group.
Mentions: To eliminate the possibility that the inhibitory action of miRNA mimics on catalase expression in presence of H2O2 was due to the toxic effect of H2O2, we measured the cellular viability at 24 h and found no significant changes in the cells transfected with either mimics (20 nM) or antagomirs (50 nM) of miR-30b, when compared to negative control (NC). H2O2 alone and together with miR-30b mimics significantly (p<0.05) reduced the cell viability, as compared to NC. H2O2 alone also showed a significant difference in cell viability when compared with mimics (p = 0.008) and antagomirs (p<0.002). However, miR-30b antagomirs significantly rescued H2O2-mediated cell death, as compared to cells treated with H2O2 alone (p = 0.004) or H2O2 with the mimics (p = 0.002) [Figure 3].

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