Ling Wang1, Wei-Xian Liu1, Xiong-Gao Huang2. 1. Department of Ophthalmology, the First Affiliated Hospital of Hainan Medical University, Haikou 570102, PR China. 2. Department of Ophthalmology, the First Affiliated Hospital of Hainan Medical University, Haikou 570102, PR China. Electronic address: hxg_eye@163.com.
Abstract
BACKGROUND: Diabetic retinopathy (DR) is a major inducer of blindness and visual impairment. As a critical cause for DR, hyperglycaemia is able to trigger multiple biochemical alterations. MiRNAs, which contain various functions, can effectively regulate blood glucose levels. This research aims to confirm the roles of miRNA-199a-3p in the progression of angiogenesis in an in vitro model of DR. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) was carried to determine the expression levels of miR-199a-3p and VEGF in both hRMECs and APRE-19 cells. The luciferase reporter assay was used to study the interaction between miR-199a-3p and VEGF. Western blot assay was conducted to examine the expression levels of VEGF and the PI3K/AKT signalling pathway. The cell proliferation capacity was detected via the CCK-8 test. The impact of miR-199a-3p on migration was determined using Transwell and wound healing assays. A Matrigel tube formation assay was employed to determine the vascular formation of hRMECs. Flow cytometry was used to determine cell apoptosis in the presence of LY294002 as a PI3K inhibitor. RESULTS: Our results showed that high glucose (HG) decreased the relative expression level of miR-199a-3p but increased VEGF expression in hRMECs and APRE-19 cells. MiR-199a-3p inhibitor augmented cell growth, migration and angiogenesis of hRMECs. Moreover, upregulation of miR-199a-3p evidently alleviated the increases in cell proliferation, migration and angiogenesis caused by HG. In addition, the luciferase reporter assay indicated that miR-199a-3p directly targeted VEGF. The overexpression of miR-199a-3p obviously restrained the HG-stimulated PI3K/AKT signalling pathway and angiogenesis, which could be further inhibited by LY294002. Moreover, LY294002 could slightly ameliorate the miR-199a-3p inhibitor-stimulated PI3K/AKT signalling pathway and angiogenesis. CONCLUSION: MiR-199a-3p upregulation ameliorated HG-stimulated angiogenesis of hRMECs by modulating the PI3K/AKT pathway through inhibiting VEGF. Although retinal neovascularization in vivo has not been studied, these in vitro findings provide more evidence for the role of miR-199a-3p upregulation against HG-induced angiogenesis.
BACKGROUND:Diabetic retinopathy (DR) is a major inducer of blindness and visual impairment. As a critical cause for DR, hyperglycaemia is able to trigger multiple biochemical alterations. MiRNAs, which contain various functions, can effectively regulate blood glucose levels. This research aims to confirm the roles of miRNA-199a-3p in the progression of angiogenesis in an in vitro model of DR. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) was carried to determine the expression levels of miR-199a-3p and VEGF in both hRMECs and APRE-19 cells. The luciferase reporter assay was used to study the interaction between miR-199a-3p and VEGF. Western blot assay was conducted to examine the expression levels of VEGF and the PI3K/AKT signalling pathway. The cell proliferation capacity was detected via the CCK-8 test. The impact of miR-199a-3p on migration was determined using Transwell and wound healing assays. A Matrigel tube formation assay was employed to determine the vascular formation of hRMECs. Flow cytometry was used to determine cell apoptosis in the presence of LY294002 as a PI3K inhibitor. RESULTS: Our results showed that high glucose (HG) decreased the relative expression level of miR-199a-3p but increased VEGF expression in hRMECs and APRE-19 cells. MiR-199a-3p inhibitor augmented cell growth, migration and angiogenesis of hRMECs. Moreover, upregulation of miR-199a-3p evidently alleviated the increases in cell proliferation, migration and angiogenesis caused by HG. In addition, the luciferase reporter assay indicated that miR-199a-3p directly targeted VEGF. The overexpression of miR-199a-3p obviously restrained the HG-stimulated PI3K/AKT signalling pathway and angiogenesis, which could be further inhibited by LY294002. Moreover, LY294002 could slightly ameliorate the miR-199a-3p inhibitor-stimulated PI3K/AKT signalling pathway and angiogenesis. CONCLUSION:MiR-199a-3p upregulation ameliorated HG-stimulated angiogenesis of hRMECs by modulating the PI3K/AKT pathway through inhibiting VEGF. Although retinal neovascularization in vivo has not been studied, these in vitro findings provide more evidence for the role of miR-199a-3p upregulation against HG-induced angiogenesis.
Authors: Przemysław A Stempor; Dror Avni; Raya Leibowitz; Yechezkel Sidi; Maria Stępień; Tomasz Dzieciątkowski; Paula Dobosz Journal: Int J Mol Sci Date: 2021-03-04 Impact factor: 5.923