Limits...
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.

Show MeSH

Related in: MedlinePlus

miR-30b interacts with the human catalase 3′UTR.ARPE-19 cells were co-transfected with wild-type (WT, Figure 5A) or mutant (mut, Figure 5B) catalase 3′-UTR s and 20 nM scrambled miRNA (NC), 20 nM mimics, or 50 nM antagomirs using Lipofectamine™ 2000 transfection reagent. Cells were harvested 24 h post-transfection for measuring luciferase activities by the dual luciferase reporter assay system, as described in ‘Materials and Methods’. The Firefly luciferase activity was normalized with the Renilla luciferase activity. The levels of luciferase activity of miR-30b mimics and miR-30b antagomirs groups were compared with those of NC-transfected cells; NC values were set to 1. Values are the means ± SEM of relative luciferase activity over NC after normalization to the Renilla luciferase activity from two independent experiments in triplicate. Sequences of wild-type or mutant (3 bp mutation within the seed region) target site for miR-30b in catalase 3′-UTR are shown above the figure. The levels of significance between the groups are shown inside the figure (5A).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3412823&req=5

pone-0042542-g005: miR-30b interacts with the human catalase 3′UTR.ARPE-19 cells were co-transfected with wild-type (WT, Figure 5A) or mutant (mut, Figure 5B) catalase 3′-UTR s and 20 nM scrambled miRNA (NC), 20 nM mimics, or 50 nM antagomirs using Lipofectamine™ 2000 transfection reagent. Cells were harvested 24 h post-transfection for measuring luciferase activities by the dual luciferase reporter assay system, as described in ‘Materials and Methods’. The Firefly luciferase activity was normalized with the Renilla luciferase activity. The levels of luciferase activity of miR-30b mimics and miR-30b antagomirs groups were compared with those of NC-transfected cells; NC values were set to 1. Values are the means ± SEM of relative luciferase activity over NC after normalization to the Renilla luciferase activity from two independent experiments in triplicate. Sequences of wild-type or mutant (3 bp mutation within the seed region) target site for miR-30b in catalase 3′-UTR are shown above the figure. The levels of significance between the groups are shown inside the figure (5A).

Mentions: To experimentally validate the computational data, a pmiR-GLO luciferase construct with the catalase 3′-UTR was generated. The purified gel product of catalase 3′-UTR was cloned into the cloning site downstream of the luciferase gene as mentioned in ‘Materials and Methods’. A mutant version of pmir-GLO-catalase-3′-UTR-mut with 3 bp mutation within the seed region (Figure 5B) was also generated. A significant decrease (p = 0.004) in relative luciferase activity was observed when pmir-GLO-catalase-3′-UTR was co-transfected with miR-30b mimics as compared with the scrambled miRNAs (NC,Figure 5A), and the miR-30b mimics-mediated suppression was abolished by the mutation of the 3′-UTR miR-30b binding site, which disrupts the interaction between miR-30b and the catalase-3′-UTR (Figure 5B). miR-30b antagomirs not only restored the wild-type 3′-UTR-modulated luciferase activity, but also significantly increased its activity compared with the NC (p = 0.002) and miR-30b mimics (p = 0.001) (Figure 5A).


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)

miR-30b interacts with the human catalase 3′UTR.ARPE-19 cells were co-transfected with wild-type (WT, Figure 5A) or mutant (mut, Figure 5B) catalase 3′-UTR s and 20 nM scrambled miRNA (NC), 20 nM mimics, or 50 nM antagomirs using Lipofectamine™ 2000 transfection reagent. Cells were harvested 24 h post-transfection for measuring luciferase activities by the dual luciferase reporter assay system, as described in ‘Materials and Methods’. The Firefly luciferase activity was normalized with the Renilla luciferase activity. The levels of luciferase activity of miR-30b mimics and miR-30b antagomirs groups were compared with those of NC-transfected cells; NC values were set to 1. Values are the means ± SEM of relative luciferase activity over NC after normalization to the Renilla luciferase activity from two independent experiments in triplicate. Sequences of wild-type or mutant (3 bp mutation within the seed region) target site for miR-30b in catalase 3′-UTR are shown above the figure. The levels of significance between the groups are shown inside the figure (5A).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0042542-g005: miR-30b interacts with the human catalase 3′UTR.ARPE-19 cells were co-transfected with wild-type (WT, Figure 5A) or mutant (mut, Figure 5B) catalase 3′-UTR s and 20 nM scrambled miRNA (NC), 20 nM mimics, or 50 nM antagomirs using Lipofectamine™ 2000 transfection reagent. Cells were harvested 24 h post-transfection for measuring luciferase activities by the dual luciferase reporter assay system, as described in ‘Materials and Methods’. The Firefly luciferase activity was normalized with the Renilla luciferase activity. The levels of luciferase activity of miR-30b mimics and miR-30b antagomirs groups were compared with those of NC-transfected cells; NC values were set to 1. Values are the means ± SEM of relative luciferase activity over NC after normalization to the Renilla luciferase activity from two independent experiments in triplicate. Sequences of wild-type or mutant (3 bp mutation within the seed region) target site for miR-30b in catalase 3′-UTR are shown above the figure. The levels of significance between the groups are shown inside the figure (5A).
Mentions: To experimentally validate the computational data, a pmiR-GLO luciferase construct with the catalase 3′-UTR was generated. The purified gel product of catalase 3′-UTR was cloned into the cloning site downstream of the luciferase gene as mentioned in ‘Materials and Methods’. A mutant version of pmir-GLO-catalase-3′-UTR-mut with 3 bp mutation within the seed region (Figure 5B) was also generated. A significant decrease (p = 0.004) in relative luciferase activity was observed when pmir-GLO-catalase-3′-UTR was co-transfected with miR-30b mimics as compared with the scrambled miRNAs (NC,Figure 5A), and the miR-30b mimics-mediated suppression was abolished by the mutation of the 3′-UTR miR-30b binding site, which disrupts the interaction between miR-30b and the catalase-3′-UTR (Figure 5B). miR-30b antagomirs not only restored the wild-type 3′-UTR-modulated luciferase activity, but also significantly increased its activity compared with the NC (p = 0.002) and miR-30b mimics (p = 0.001) (Figure 5A).

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