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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

Effects of miR-30b mimics and antagomirs on catalase protein expression in ARPE-19 cells.Cells transfected for 24 h with NC, miR-30b mimics, or miR-30b antagomirs, were lysed and subjected to western blotting following the protocol mentioned in ‘Materials and Methods’. The PVDF membrane was immunoblotted with anti-catalase and anti-μ-actin antibodies. β-Actin was used as a loading control. Data reported here are representative of the experiment performed in triplicate. The ratio of band intensity is relative to that of β-actin. The band intensity was measured by using ImageJ software (see ‘Materials and Methods’). Values are presented as mean ± SEM; n = 3. *p<0.05 vs. control, # p<0.05 vs. miR-30b mimics.
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pone-0042542-g006: Effects of miR-30b mimics and antagomirs on catalase protein expression in ARPE-19 cells.Cells transfected for 24 h with NC, miR-30b mimics, or miR-30b antagomirs, were lysed and subjected to western blotting following the protocol mentioned in ‘Materials and Methods’. The PVDF membrane was immunoblotted with anti-catalase and anti-μ-actin antibodies. β-Actin was used as a loading control. Data reported here are representative of the experiment performed in triplicate. The ratio of band intensity is relative to that of β-actin. The band intensity was measured by using ImageJ software (see ‘Materials and Methods’). Values are presented as mean ± SEM; n = 3. *p<0.05 vs. control, # p<0.05 vs. miR-30b mimics.

Mentions: The Western blotting analyses further confirmed the luciferase assay results. Here, transfection with the mimics of miR-30b resulted in decrease of catalase protein expression (p<0.05) when compared with the control, whereas the antagomirs of miR-30b protected the miR-30b mimics-mediated inhibition of catalase expression (p<0.05) even in presence of miR-30b mimics (p<0.05) (Figure 6).


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)

Effects of miR-30b mimics and antagomirs on catalase protein expression in ARPE-19 cells.Cells transfected for 24 h with NC, miR-30b mimics, or miR-30b antagomirs, were lysed and subjected to western blotting following the protocol mentioned in ‘Materials and Methods’. The PVDF membrane was immunoblotted with anti-catalase and anti-μ-actin antibodies. β-Actin was used as a loading control. Data reported here are representative of the experiment performed in triplicate. The ratio of band intensity is relative to that of β-actin. The band intensity was measured by using ImageJ software (see ‘Materials and Methods’). Values are presented as mean ± SEM; n = 3. *p<0.05 vs. control, # p<0.05 vs. miR-30b mimics.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0042542-g006: Effects of miR-30b mimics and antagomirs on catalase protein expression in ARPE-19 cells.Cells transfected for 24 h with NC, miR-30b mimics, or miR-30b antagomirs, were lysed and subjected to western blotting following the protocol mentioned in ‘Materials and Methods’. The PVDF membrane was immunoblotted with anti-catalase and anti-μ-actin antibodies. β-Actin was used as a loading control. Data reported here are representative of the experiment performed in triplicate. The ratio of band intensity is relative to that of β-actin. The band intensity was measured by using ImageJ software (see ‘Materials and Methods’). Values are presented as mean ± SEM; n = 3. *p<0.05 vs. control, # p<0.05 vs. miR-30b mimics.
Mentions: The Western blotting analyses further confirmed the luciferase assay results. Here, transfection with the mimics of miR-30b resulted in decrease of catalase protein expression (p<0.05) when compared with the control, whereas the antagomirs of miR-30b protected the miR-30b mimics-mediated inhibition of catalase expression (p<0.05) even in presence of miR-30b mimics (p<0.05) (Figure 6).

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