microRNA-383 mediates high glucose-induced oxidative stress and apoptosis in retinal pigment epithelial cells by repressing peroxiredoxin 3

Abstract

Hyperglycemia-mediated damage to retinal pigment epithelial (RPE) cells plays a central role in the pathogenesis of diabetic retinopathy. Dysregulation of microRNA (miR)-383 modulates pancreatic beta cell survival in diabetes; however, its role in diabetic retinopathy remains unclear. In this study, we examined the expression of miR-383 in ARPE-19 human RPE cell lines after high glucose treatment and investigated its functions in high glucose-induced reactive oxygen species (ROS) generation and apoptotic responses. The downstream target gene that mediated the action of miR-383 was functionally characterized. It was found that high glucose induced a 2.4-fold increase in miR-383 abundance, compared to ARPE-19 cells treated with normal glucose. Overexpression of miR-383 inhibited cell viability and promoted apoptosis and ROS formation in ARPE-19 cells, which was coupled with deregulation of Bcl-2 and Bax. Peroxiredoxin 3 (PRDX3) expression was repressed by miR-383 in ARPE-19 cells. Restoration of PRDX3 counteracted miR-383-induced ROS generation and apoptosis, while silencing of PRDX3 phenocopied the detrimental effects of miR-383 on ARPE-19 cells. Delivery of anti-miR-383 inhibitors led to an increase of PRDX3 expression and prevented high glucose-elicited ROS formation and apoptosis in ARPE-19 cells. Overall, miR-383 upregulation accounts for high glucose-induced oxidative stress and apoptosis in RPE cells by repressing PRDX3 expression. Targeting miR-383 may have therapeutic potential in the treatment of diabetic retinopathy.

Keywords: Apoptosis diabetic retinopathy; microRNA; oxidative stress; target gene.

Figure 1

Upregulation of miR-383 in response to high glucose triggers apoptosis in ARPE-19 cells. A. qRT-PCR analysis of miR-383 levels in ARPE-19 cells exposed to normal or high glucose. High glucose treatment increased the expression of miR-383. *P < 0.05 vs. cells under normal glucose conditions. B. ARPE-19 cells were transfected with control miRNA (control) or miR-383 mimic and tested for viability at 48 h posttransfection by MTT assay. miR-383 overexpression caused a reduction of cell viability. C. Flow cytometric analysis of apoptosis after Annexin-V/PI staining. Ectopic expression of miR-383 induced apoptosis in ARPE-19 cells. Left, representative dot plots of apoptotic cells. D. Western blot analysis of Bcl-2 and Bax protein levels. Right, quantification of the Bax/Bcl-2 protein ratio. *P < 0.05 vs. cells transfected with control miRNA.

Figure 2

Promotion of ROS release is involved in miR-383-induced apoptosis in ARPE-19 cells. ARPE-19 cells were transfected with control miRNA or miR-383 mimic with or without pretreatment with NAC or vehicle. ROS production and apoptosis were assessed 48 h posttransfection. A. Measurement of intracellular ROS generation using the DCFH-DA probe by flow cytometry. Top, representative flow cytometric histogram of DCF fluorescent levels. Bottom, quantification of intracellular ROS levels from three independent experiments. B. Quantification of apoptotic death by flow cytometry after Annexin-V/PI staining. *P < 0.05.

Figure 3

Overexpression of PRDX3 reverses miR-383-mediated ROS formation and apoptosis. A. Western blot analysis of PRDX3 and PRDX6 protein levels in ARPE-19 cells transfected with indicated constructs. *P < 0.05 vs. control cells; #P < 0.05 vs. cells co-transfected with miR-383 mimic and PRDX3-expressing plasmid. B. Intracellular ROS generation was determined using the DCFH-DA probe by flow cytometry. C. Detection of apoptotic death by flow cytometry after Annexin-V/PI staining. D. Western blot analysis of Bax and Bcl-2 protein levels. Bottom, quantification of the Bax/Bcl-2 protein ratio. *P < 0.05.

Figure 4

PRDX3 knockdown promotes ROS formation and apoptosis. A. Western blot analysis of PRDX3 protein levels in ARPE-19 cells transfected with control siRNA or PRDX3 siRNA. B. Intracellular ROS generation was determined using the DCFH-DA probe by flow cytometry. C. Detection of apoptotic death by flow cytometry after Annexin-V/PI staining. D. Western blot analysis of Bax and Bcl-2 protein levels. Right, quantification of the Bax/Bcl-2 protein ratio. *P < 0.05 vs. cells transfected with control siRNA.

Figure 5

Targeting miR-383 blocks high glucose-induced oxidative stress and apoptosis. A. Western blot analysis of PRDX3protein levels in ARPE-19 cells transfected with control inhibitor (control) or anti-miR-383 inhibitor. B and C. ARPE-19 cells were pretransfected with control inhibitor or anti-miR-383 inhibitor 24 h before exposure to high glucose. B. Intracellular ROS formation was determined by flow cytometry. C. Detection of apoptotic death by flow cytometry after Annexin-V/PI staining. *P < 0.05.