Circular RNA-ZNF532 regulates diabetes-induced retinal pericyte degeneration and vascular dysfunction
Qin Jiang, … , Chen Zhao, Biao Yan
Qin Jiang, … , Chen Zhao, Biao Yan
Published April 28, 2020
Citation Information: J Clin Invest. 2020;130(7):3833-3847. https://doi.org/10.1172/JCI123353.
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Research Article Ophthalmology

Circular RNA-ZNF532 regulates diabetes-induced retinal pericyte degeneration and vascular dysfunction

Abstract

Diabetic retinopathy (DR) is the leading cause of blindness in working-age adults. Vascular pericyte degeneration is the predominant clinical manifestation of DR, yet the mechanism governing pericyte degeneration is poorly understood. Circular RNAs (circRNAs) play important roles in multiple biological processes and disease progression. Here, we investigated the role of circRNA in pericyte biology and diabetes-induced retinal vascular dysfunction. cZNF532 expression was upregulated in pericytes under diabetic stress, in the retinal vessels of a diabetic murine model, and in the vitreous humor of diabetic patients. cZNF532 silencing reduced the viability, proliferation, and differentiation of pericytes and suppressed the recruitment of pericytes toward endothelial cells in vitro. cZNF532 regulated pericyte biology by acting as a miR-29a-3p sponge and inducing increased expression of NG2, LOXL2, and CDK2. Knockdown of cZNF532 or overexpression of miR-29a-3p aggravated streptozotocin-induced retinal pericyte degeneration and vascular dysfunction. By contrast, overexpression of cZNF532 or inhibition of miR-29a-3p ameliorated human diabetic vitreous–induced retinal pericyte degeneration and vascular dysfunction. Collectively, these data identify a circRNA-mediated mechanism that coordinates pericyte biology and vascular homeostasis in DR. Induction of cZNF532 or antagonism of miR-29a-3p is an exploitable therapeutic approach for the treatment of DR.

Authors

Qin Jiang, Chang Liu, Chao-Peng Li, Shan-Shan Xu, Mu-Di Yao, Hui-Min Ge, Ya-Nan Sun, Xiu-Miao Li, Shu-Jie Zhang, Kun Shan, Bai-Hui Liu, Jin Yao, Chen Zhao, Biao Yan

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Figure 2

cZNF532 regulates pericyte function in vitro.

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cZNF532 regulates pericyte function in vitro. (A) The expression of peri...
(A) The expression of pericyte markers, including PDGFR-β, α-SMA, desmin, and NG2, was detected by qRT-PCRs in pericytes after the transfection of scrambled (Scr) siRNA or cZNF532 siRNA1, or left untreated (Ctrl) for 24 hours and 48 hours (n = 4). (B) WT (Ctrl), cZNF532 siRNA1, or Scr siRNA–transfected pericytes were cocultured with HRVECs for 8 hours or 12 hours and then stained with NG2 (pericytes) and CD31 (HRVECs) to detect the recruitment of pericytes toward HRVECs (n = 4). The representative images at 12 hours are shown. Scale bar: 100 μm. (C) WT (Ctrl), cZNF532 siRNA1, or Scr siRNA–transfected pericytes were cocultured with HRVECs for 8 hours and 12 hours. Fluorescent solute (FITC-Dextran, 70 kDa) was added to the apical chamber and the rate of flux across the HRVEC monolayer was detected by a microplate reader. Endothelial barrier permeability was shown as relative diffusive flux change (n = 4). Average Po for the control samples at 8 hours and 12 hours was 2.78 × 10–6 cm/sec and 1.62 × 10–6 cm/sec, respectively. (D and E) Pericytes were transfected with Scr siRNA or cZNF532 siRNA1, or left untreated (Ctrl) for 24 hours or 48 hours. Cell viability was detected by MTT method (D, n = 4). Cell proliferation was detected by Ki67 staining. The representative images at 48 hours were shown (E, scale bar: 20 μm, n = 4). (F) Cell cycles of pericytes were detected by flow cytometry 48 hours after the transfection of Scr siRNA or cZNF532 siRNA1. Cell percentage in each phase was calculated by BD Cell Quest Pro software. (G and H) Pericytes were transfected with Scr siRNA or cZNF532 siRNA1, or left untreated (Ctrl), and then exposed to 30 mM glucose for 24 hours or 48 hours. Apoptotic cells were detected by caspase-3/7 activity (G, n = 4) or PI staining (H, n = 4). Representative PI staining images at 48 hours were shown (scale bar: 100 μm). All significant difference was determined by 1-way ANOVA followed by Bonferroni’s post hoc test. Error bar indicates SD. *P < 0.05.