Theranostics 2020; 10(7):3293-3307. doi:10.7150/thno.39488

Research Paper

Circular RNA-ZBTB44 regulates the development of choroidal neovascularization

Rong-mei Zhou1*, Lian-jun Shi2,3*, Kun Shan1*, Ya-nan Sun1, Shan-shan Wang5, Shu-jie Zhang1,4, Xiu-miao Li2,3, Qin Jiang2,3, Biao Yan1,4✉, Chen Zhao1,4,6✉

1. Eye Institute, Eye & ENT Hospital, Shanghai Medical College, Fudan University, Shanghai, China
2. The Fourth School of Clinical Medicine, Nanjing Medical University, Nanjing, China
3. Eye Hospital, Nanjing Medical University, Nanjing, China
4. NHC Key Laboratory of Myopia (Fudan University), Key Laboratory of Myopia, Chinese Academy of Medical Sciences, and Shanghai Key Laboratory of Visual Impairment and Restoration (Fudan University), Shanghai, China
5. Department of Ophthalmology, The First Affiliated Hospital of Nanjing Medical University, State Key Laboratory of Reproductive Medicine, Nanjing, China
6. State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
*These authors contributed equally to this work.

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Citation:
Zhou Rm, Shi Lj, Shan K, Sun Yn, Wang Ss, Zhang Sj, Li Xm, Jiang Q, Yan B, Zhao C. Circular RNA-ZBTB44 regulates the development of choroidal neovascularization. Theranostics 2020; 10(7):3293-3307. doi:10.7150/thno.39488. Available from http://www.thno.org/v10p3293.htm

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Abstract

Rationale: Choroidal neovascularization (CNV) is a major cause of severe vision loss and occurs in many ocular diseases, especially neovascular age-related macular degeneration (nAMD). Circular RNAs (circRNAs) are emerging as a new class of endogenous noncoding RNAs, which have been implicated in the regulation of endothelial cell dysfunction in diabetes mellitus and cancer. In this study, we aimed to determine the role of circRNA-ZBTB44 (cZBTB44) in the pathogenesis of CNV.

Methods: Quantitative polymerase chain reaction was conducted to detect cZBTB44 expression pattern during CNV development. Isolectin B4 staining, hematoxylin and eosin (HE) staining, and choroidal sprouting assay ex vivo were conducted to evaluate the role of cZBTB44 in the development of CNV. Endothelial cell proliferation, migration and tube formation assays were conducted to determine the role of cZBTB44 in angiogenic effect in vitro. Bioinformatics analysis, RNA immunoprecipitation assay, luciferase assay, and in vitro studies were conducted to investigate the mechanism of cZBTB44-mediated CNV development.

Results: cZBTB44 expression was significantly up-regulated in a laser-induced CNV mouse model in vivo and in endothelial cells upon hypoxia stress in vitro. cZBTB44 silencing retarded CNV development, while overexpression of cZBTB44 showed the opposite effects. The role of cZBTB44 in CNV development was confirmed in choroidal sprouting assay ex vivo. cZBTB44 silencing reduced endothelial cell viability, proliferation, migration and tube formation in vitro. cZBTB44 acted as miR-578 sponge to sequester and inhibit miR-578 activity, which led to increased expression of vascular endothelial growth factor A (VEGFA) and vascular cell adhesion molecule-1 (VCAM1). Overexpression of miR-578 mimicked cZBTB44 silencing-mediated anti-angiogenic effects in vivo and in vitro. Furthermore, dysregulated cZBTB44 expression was detected in the clinical samples of nAMD patients.

Conclusions: This study provided novel insights into the molecular pathogenesis of CNV. The cZBTB44-miR-578-VEGFA/VCAM1 axis might be a potential source of novel therapeutic targets for neovascularization-related diseases.

Keywords: choroidal neovascularization, circular RNA, cZBTB44, miR-578 sponge