MicroRNA-100 Mediates Hydrogen Peroxide-Induced Apoptosis of Human Retinal Pigment Epithelium ARPE-19 Cells

Abstract

This study investigated the regulatory role of microRNA 100 (miR-100) in hydrogen peroxide (H₂O₂)-induced apoptosis of human retinal pigment epithelial ARPE-19 cells. H₂O₂ induced oxidative cell death of cultured ARPE-19 cells was measured by cytotoxicity assay. qRT-PCR was used to quantify cytosolic and extracellular contents of miR-100. Kinase and miR-100 inhibition treatments were applied to determine the regulatory signaling pathways involved in cell death regulation. H₂O₂ dose-dependently reduced viability of ARPE-19 cells and simultaneously upregulated miR-100 levels in both cytosolic and extracellular compartments. Western blotting detection indicated that H₂O₂ elicited hyperphosphorylation of PI3K/Akt, ERK1/2, JNK, p38 MAPK, and p65 NF-κB. Further kinase inhibition experiments demonstrated that PI3K, p38 MAPK, and NF-κB activities were involved in oxidative-stress-induced miR-100 upregulation in ARPE-19 cells, while blockade of PI3K, JNK, and NF-κB signaling significantly attenuated the oxidative cell death. Intriguingly, MiR-100 antagomir treatment exerted a cytoprotective effect against the H₂O₂-induced oxidative cell death through attenuating the oxidation-induced AMPK hyperphosphorylation, restoring cellular mTOR and p62/SQSTM1 levels and upregulating heme oxygenase-1 expression. These findings support that miR-100 at least in part mediates H₂O₂-induced cell death of ARPE-19 cells and can be regarded as a preventive and therapeutic target for retinal degenerative disease.

Keywords: heme oxygenase-1; mTOR; microRNA biosynthesis; oxidative stress; signal transduction.

Figure 1

Hydrogen peroxide treatment induces miR-100 biosynthesis in human ARPE-19 cells. (A) ARPE-19 cells were treated with H₂O₂ at indicated doses for 24 h and subjected to cellular viability assay. (B,C) Alternatively, biosynthesis and release of miRNA was measured by isolating miRNA from lysates and conditioned media, followed by miRNA-100 quantification using RT-qPCR. All data are shown in mean ± SD from three independent experiments. * p < 0.05 vs. zero control by t-Test. MC, medium control; ND, not detectable.

Figure 2

Characterization of hydrogen peroxide-driven signaling kinetics in human ARPE-19 cells. (A) ARPE-19 cells were treated with 225 μM of H₂O₂ for the indicated time. Cellular protein lysates were subjected to western blotting detection for total and phosphorylated kinase proteins. Densitometric analysis was used to quantify phosphorylation levels of Akt (B), ERK1/2 (C), JNK (D), p38 MAPK (E), and NF-κB (F). Data are expressed as mean values from three independent experiments.

Figure 3

Involvement of hydrogen peroxide-activated signaling in induced miR-100 biosynthesis in human ARPE-19 cells. ARPE-19 cells were pretreated with kinase-specific inhibitors for 1 h, including the PI3K inhibitor LY294002 (LY, 10 µM), MEK1 inhibitor PD98059 (PD, 10 µM), p38 inhibitor SB203580 (SB, 20 µM), JNK inhibitor SP600125 (SP, 20 µM), NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC, 50 µM), or equivalent dimethyl sulfoxide (DMSO) as solvent control, followed by H₂O₂ exposure at 225 µM. After 24 h of treatment, lysates (A) and conditioned media (B) were subjected to miRNA isolation and subsequent RT-qPCR detection for miRNA-100 levels. Semi-quantitative mRNA levels are shown as an induction fold of negative control (NC) in mean ± SD from three independent experiments. * p < 0.05 vs. NC group; # p < 0.05 vs. DMSO groups by ANOVA.

Figure 4

Involvement of hydrogen peroxide-activated signaling in its cytotoxic effect on human ARPE-19 cells. ARPE-19 cells were pretreated with kinase-specific inhibitors for 1 h, including the PI3K inhibitor LY294002 (LY, 10 µM), MEK1 inhibitor PD98059 (PD, 10 µM), p38 inhibitor SB203580 (SB, 20 µM), JNK inhibitor SP600125 (SP, 20 µM), NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC, 50 µM), or equivalent dimethyl sulfoxide (DMSO) as solvent control, followed by transient H₂O₂ exposure at 225 µM for 2 h. After another 22-h incubation, the treated cells were subjected to cytotoxicity assay. Cell viability is expressed as a percentage of negative control (NC) in mean ± SD from three independent experiments. * p < 0.05 vs. NC group; # p < 0.05 vs. DMSO groups by ANOVA.

Figure 5

Mitigation of hydrogen peroxide-induced apoptosis of human ARPE-19 cells by miR-100 antagonism. ARPE-19 cells were transfected with either a 200 nM miR-100 antagonist or control oligonucleotides for 24 h followed by H₂O₂ exposure at 225 µM for another 24 h. (A) Cellular lysates and conditioned media were collected for miRNA isolation and miR-100 detection by RT-qPCR. ND, not detectable. (B) Cytotoxicity assay was used to determine the viability of the treated cells. Alternatively, the treated cells were fixed and subjected to fluorescent TUNEL staining (C) and subsequent analysis on TUNEL staining positivity (D). Data are expressed as mean ± SD from three independent experiments. * p < 0.05 vs. control or between indicated groups, by ANOVA. Scale bar = 50 µm. +/− indicates presence/absence of reagents.

Figure 6

Effects of miR-100 inhibition on AMPK/mTOR signaling and heme oxygenase 1 (HO-1) expression in human ARPE-19 cells. ARPE-19 cells were transfected with either an miR-100 inhibitor or negative miRNA control nucleotides for 24 h followed by H₂O₂ exposure at 225 μM for another 24 h. After treatments, cellular protein lysates were collected for western blotting detection (A) and subsequent densitometry analysis for p-AMPK (B), total mTOR (C), HO-1 (D), and p62 levels (E). Data are expressed as mean ± SD from three independent experiments. * p < 0.05 between indicated groups by ANOVA. +/− indicates presence/absence of reagents.

Figure 7

Effects of miR-100 inhibition on cellular expression and distribution of p62 in human ARPE-19 cells under oxidative stress. ARPE-19 cells were transfected with either miR-100 inhibitor or miRNA control nucleotides for 24 h followed by H₂O₂ exposure at 225 μM for another 24 h. Afterward, the cells were fixed and subjected to immunofluorescent staining. Note that miR-100 inhibition restored autophagy-associated perinuclear granules in the H₂O₂-exposed cells. Scale bar = 20 μm.

Figure 8

A hypothetical scheme showing the network of oxidative-stress-induced signal pathways, miR-100 biosynthesis, and their roles in the oxidative cell death of retinal pigment epithelial (RPE) cells. The study findings demonstrated that hydrogen peroxide (H₂O₂)-mimicked oxidative stress may activate signaling pathways including the hyperphosphorylation of Akt, JNK, p38 MAPK, ERK, and NF-κB. The mediator activities of PI3K/Akt, p38 MAPK, and NF-κB contribute to endogenous miR-100 expression, thereby leading to apoptotic cell death. The NF-κB signal mediator very likely lies downstream of the Akt and JNK axes, which awaits further elucidation. The PI3K/Akt/mTOR axis is known to mediate cell survival signals. Mechanistically, miR-100 antagomir treatment not only attenuates oxidative stress-related AMPK phosphorylation but also restores cellular mTOR and p62 levels and upregulates antioxidant heme oxygenase-1 (HO-1) expression, thereby protecting RPE cells against oxidative cell death. Arrows, stimulatory; T lines, inhibitory; Solid lines, new findings of this study; Dashed line, previously identified mechanism; Question marks, unknown mechanisms.