Kun Shan1, Chang Liu1, Bai-Hui Liu1, Xue Chen1, Rui Dong1, Xin Liu1, Yang-Yang Zhang1, Ban Liu1, Shu-Jie Zhang1, Jia-Jian Wang1, Sheng-Hai Zhang1, Ji-Hong Wu1, Chen Zhao2, Biao Yan2. 1. From Eye Institute, Eye and ENT Hospital, Shanghai Medical College, Fudan University, China (K.S., C.L., X.L., S.-J.Z., J.-J.W., S.-H.Z., J.-H.W., C.Z., B.Y.); Fourth School of Clinical Medicine, Nanjing Medical University, China (C.L.); Department of Pediatric Surgery, Children's Hospital of Fudan University, Shanghai, China (B.-H.L., R.D.); Department of Ophthalmology, First Affiliated Hospital of Nanjing Medical University, China (X.C.); Department of Cardiovascular Surgery, Shanghai East Hospital, Tongji University School of Medicine, China (Y.-Y.Z.); Department of Cardiology, Shanghai Tenth People's Hospital, Tongji University School of Medicine, China (B.L.); and Shanghai Key Laboratory of Visual Impairment and Restoration, China (C.Z., B.Y.). 2. From Eye Institute, Eye and ENT Hospital, Shanghai Medical College, Fudan University, China (K.S., C.L., X.L., S.-J.Z., J.-J.W., S.-H.Z., J.-H.W., C.Z., B.Y.); Fourth School of Clinical Medicine, Nanjing Medical University, China (C.L.); Department of Pediatric Surgery, Children's Hospital of Fudan University, Shanghai, China (B.-H.L., R.D.); Department of Ophthalmology, First Affiliated Hospital of Nanjing Medical University, China (X.C.); Department of Cardiovascular Surgery, Shanghai East Hospital, Tongji University School of Medicine, China (Y.-Y.Z.); Department of Cardiology, Shanghai Tenth People's Hospital, Tongji University School of Medicine, China (B.L.); and Shanghai Key Laboratory of Visual Impairment and Restoration, China (C.Z., B.Y.). yanbiao1982@hotmail.com dr_zhaochen@163.com.
Abstract
BACKGROUND: The vascular complications of diabetes mellitus are the major causes of morbidity and mortality among people with diabetes. Circular RNAs are a class of endogenous noncoding RNAs that regulate gene expression in eukaryotes. In this study, we investigated the role of circular RNA in retinal vascular dysfunction induced by diabetes mellitus. METHODS: Quantitative polymerase chain reactions, Sanger sequencing, and Northern blots were conducted to detect circular HIPK3 (circHIPK3) expression pattern on diabetes mellitus-related stresses. MTT (3-[4,5-dimethythiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assays, EdU (5-ethynyl-2'-deoxyuridine) incorporation assays, Transwell migration assays, and Matrigel assays were conducted to detect the role of circHIPK3 in retinal endothelial cell function in vitro. Retinal trypsin digestion, vascular permeability assays, and ELISA assays were conducted to detect the role of circHIPK3 in retinal vascular dysfunction in vivo. Bioinformatics analysis, luciferase activity assays, RNA pull-down assays, and in vitro studies were conducted to reveal the mechanism of circHIPK3-mediated retinal vascular dysfunction. RESULTS: circHIPK3 expression was significantly upregulated in diabetic retinas and retinal endothelial cells following stressors related to diabetes mellitus. circHIPK3 silencing or overexpressing circHIPK3 changed retinal endothelial cell viability, proliferation, migration, and tube formation in vitro. circHIPK3 silencing in vivo alleviated retinal vascular dysfunction, as shown by decreased retinal acellular capillaries, vascular leakage, and inflammation. circHIPK3 acted as an endogenous miR-30a-3p sponge to sequester and inhibit miR-30a-3p activity, which led to increased vascular endothelial growth factor-C, FZD4, and WNT2 expression. Ectopic expression of miR-30a-3p mimicked the effect of circHIPK3 silencing on vascular endothelial phenotypes in vivo and in vitro. CONCLUSIONS: The circular RNA circHIPK3 plays a role in diabetic retinopathy by blocking miR-30a function, leading to increased endothelial proliferation and vascular dysfunction. These data suggest that circular RNA is a potential target to control diabetic proliferative retinopathy.
BACKGROUND: The vascular complications of diabetes mellitus are the major causes of morbidity and mortality among people with diabetes. Circular RNAs are a class of endogenous noncoding RNAs that regulate gene expression in eukaryotes. In this study, we investigated the role of circular RNA in retinal vascular dysfunction induced by diabetes mellitus. METHODS: Quantitative polymerase chain reactions, Sanger sequencing, and Northern blots were conducted to detect circular HIPK3 (circHIPK3) expression pattern on diabetes mellitus-related stresses. MTT (3-[4,5-dimethythiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assays, EdU (5-ethynyl-2'-deoxyuridine) incorporation assays, Transwell migration assays, and Matrigel assays were conducted to detect the role of circHIPK3 in retinal endothelial cell function in vitro. Retinal trypsin digestion, vascular permeability assays, and ELISA assays were conducted to detect the role of circHIPK3 in retinal vascular dysfunction in vivo. Bioinformatics analysis, luciferase activity assays, RNA pull-down assays, and in vitro studies were conducted to reveal the mechanism of circHIPK3-mediated retinal vascular dysfunction. RESULTS: circHIPK3 expression was significantly upregulated in diabetic retinas and retinal endothelial cells following stressors related to diabetes mellitus. circHIPK3 silencing or overexpressing circHIPK3 changed retinal endothelial cell viability, proliferation, migration, and tube formation in vitro. circHIPK3 silencing in vivo alleviated retinal vascular dysfunction, as shown by decreased retinal acellular capillaries, vascular leakage, and inflammation. circHIPK3 acted as an endogenous miR-30a-3p sponge to sequester and inhibit miR-30a-3p activity, which led to increased vascular endothelial growth factor-C, FZD4, and WNT2 expression. Ectopic expression of miR-30a-3p mimicked the effect of circHIPK3 silencing on vascular endothelial phenotypes in vivo and in vitro. CONCLUSIONS: The circular RNA circHIPK3 plays a role in diabetic retinopathy by blocking miR-30a function, leading to increased endothelial proliferation and vascular dysfunction. These data suggest that circular RNA is a potential target to control diabetic proliferative retinopathy.
Authors: Xiuyuan Chen; Rui Mao; Wenmei Su; Xia Yang; Qianqian Geng; Chunfang Guo; Zhuwen Wang; Jun Wang; Laura A Kresty; David G Beer; Andrew C Chang; Guoan Chen Journal: Autophagy Date: 2019-06-28 Impact factor: 16.016