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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

Cell viability (A) and protein carbonylation (B) in H2O2-treated ARPE-19.ARPE-19 cells were treated with various doses of H2O2 for 18 h and assessed for cell viability (A) and protein carbonylation (B). Data were analyzed using analysis of variance (ANOVA) with Student–Newman–Keuls multiple comparison tests. Data are expressed as a percentage of the untreated control. n = 5, mean ± SEM, *p = 0.002 vs. control, **p<0.001 vs. control, #p<0.001 vs. 100 µM.
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pone-0042542-g002: Cell viability (A) and protein carbonylation (B) in H2O2-treated ARPE-19.ARPE-19 cells were treated with various doses of H2O2 for 18 h and assessed for cell viability (A) and protein carbonylation (B). Data were analyzed using analysis of variance (ANOVA) with Student–Newman–Keuls multiple comparison tests. Data are expressed as a percentage of the untreated control. n = 5, mean ± SEM, *p = 0.002 vs. control, **p<0.001 vs. control, #p<0.001 vs. 100 µM.

Mentions: Addition of H2O2 to the medium of cultured cells has been used as a technique to assess aspects of the oxidative defense system in RPE cells. Before examining the effect of H2O2-mediated oxidative stress on catalase and miRNA expression in ARPE-19 cells, the viability of ARPE-19 cells was determined using the CellTiter-Blue assay by exposing the cells to 25, 50, 100, 200, and 500 µM H2O2 for 18 h. Cells treated with vehicle (0 µM H2O2) were maintained as control. The CellTiter-Blue viability assay showed that H2O2 induced a gradual reduction in CellTiter-Blue values in a dose dependent manner after 18 h treatment under our experimental conditions as mentioned in ‘Materials and Methods’. H2O2 at low concentrations (<100 µmol/L) gradually reduced cell viability, but at higher concentrations (>100 µmol/L), a significant reduction (p<0.05) of cell viability was detected, as compared to control cells treated with 0 µM H2O2 (Figure 2A). For example, the CellTiter-Blue value was reduced by 12.5% (p<0.05 vs control) and 12.75% (p<0.05 vs. control) of untreated control cells after 18 h exposure to 200 µM and 500 µM H2O2, respectively. The CellTiter-Blue value observed at the 500 µM H2O2 concentration was not significantly different from that observed at the 100 µM (p = 0.802) or 200 µM (p = 0.938) H2O2 treatments. Protein carbonyls are the most widely studied markers of protein oxidation and are frequently used as markers of oxidative stress [29]. To determine the effect of H2O2-mediated oxidative stress on protein carbonylation, ARPE-19 cells were treated with various doses of H2O2 for 18 h. Protein carbonyls were measured by colorimetric assay in total protein that was isolated from H2O2-treated cells. Compared to untreated cells, a significant increase in protein carbonyls was observed in cells treated with H2O2 (100 µM–500 µM), suggesting that exposure to H2O2 -mediated oxidative stress produced damage to cell proteins (Figure 2B). In the rest of our experiments, 200 µM concentration of H2O2 was used to assess the H2O2-mediated effect on gene expression.


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)

Cell viability (A) and protein carbonylation (B) in H2O2-treated ARPE-19.ARPE-19 cells were treated with various doses of H2O2 for 18 h and assessed for cell viability (A) and protein carbonylation (B). Data were analyzed using analysis of variance (ANOVA) with Student–Newman–Keuls multiple comparison tests. Data are expressed as a percentage of the untreated control. n = 5, mean ± SEM, *p = 0.002 vs. control, **p<0.001 vs. control, #p<0.001 vs. 100 µM.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0042542-g002: Cell viability (A) and protein carbonylation (B) in H2O2-treated ARPE-19.ARPE-19 cells were treated with various doses of H2O2 for 18 h and assessed for cell viability (A) and protein carbonylation (B). Data were analyzed using analysis of variance (ANOVA) with Student–Newman–Keuls multiple comparison tests. Data are expressed as a percentage of the untreated control. n = 5, mean ± SEM, *p = 0.002 vs. control, **p<0.001 vs. control, #p<0.001 vs. 100 µM.
Mentions: Addition of H2O2 to the medium of cultured cells has been used as a technique to assess aspects of the oxidative defense system in RPE cells. Before examining the effect of H2O2-mediated oxidative stress on catalase and miRNA expression in ARPE-19 cells, the viability of ARPE-19 cells was determined using the CellTiter-Blue assay by exposing the cells to 25, 50, 100, 200, and 500 µM H2O2 for 18 h. Cells treated with vehicle (0 µM H2O2) were maintained as control. The CellTiter-Blue viability assay showed that H2O2 induced a gradual reduction in CellTiter-Blue values in a dose dependent manner after 18 h treatment under our experimental conditions as mentioned in ‘Materials and Methods’. H2O2 at low concentrations (<100 µmol/L) gradually reduced cell viability, but at higher concentrations (>100 µmol/L), a significant reduction (p<0.05) of cell viability was detected, as compared to control cells treated with 0 µM H2O2 (Figure 2A). For example, the CellTiter-Blue value was reduced by 12.5% (p<0.05 vs control) and 12.75% (p<0.05 vs. control) of untreated control cells after 18 h exposure to 200 µM and 500 µM H2O2, respectively. The CellTiter-Blue value observed at the 500 µM H2O2 concentration was not significantly different from that observed at the 100 µM (p = 0.802) or 200 µM (p = 0.938) H2O2 treatments. Protein carbonyls are the most widely studied markers of protein oxidation and are frequently used as markers of oxidative stress [29]. To determine the effect of H2O2-mediated oxidative stress on protein carbonylation, ARPE-19 cells were treated with various doses of H2O2 for 18 h. Protein carbonyls were measured by colorimetric assay in total protein that was isolated from H2O2-treated cells. Compared to untreated cells, a significant increase in protein carbonyls was observed in cells treated with H2O2 (100 µM–500 µM), suggesting that exposure to H2O2 -mediated oxidative stress produced damage to cell proteins (Figure 2B). In the rest of our experiments, 200 µM concentration of H2O2 was used to assess the H2O2-mediated effect on gene expression.

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