Department of Ophthalmology, the First Affiliated Hospital of Hainan Medical University, Haikou 570102, PR China.
Department of Ophthalmology, the First Affiliated Hospital of Hainan Medical University, Haikou 570102, PR China.
Exp Mol Pathol. 2020 Oct;116:104488. doi: 10.1016/j.yexmp.2020.104488. Epub 2020 Jul 2.
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.
背景：糖尿病视网膜病变（DR）是失明和视力障碍的主要原因。高血糖是 DR 的一个关键病因，可引发多种生化改变。miRNA 具有多种功能，可有效调节血糖水平。本研究旨在通过体外 DR 模型确认 miRNA-199a-3p 在血管生成进展中的作用。
方法：采用实时定量聚合酶链反应（qRT-PCR）测定 hRMECs 和 APRE-19 细胞中 miR-199a-3p 和 VEGF 的表达水平。利用荧光素酶报告基因实验研究 miR-199a-3p 与 VEGF 的相互作用。采用 Western blot 检测 VEGF 和 PI3K/AKT 信号通路的表达水平。通过 CCK-8 试验检测细胞增殖能力。通过 Transwell 和划痕愈合试验测定 miR-199a-3p 对迁移的影响。采用 Matrigel 管形成试验测定 hRMECs 的血管形成能力。使用 PI3K 抑制剂 LY294002 测定细胞凋亡情况。
结果：我们的结果表明，高糖（HG）降低了 hRMECs 和 APRE-19 细胞中 miR-199a-3p 的相对表达水平，但增加了 VEGF 的表达。miR-199a-3p 抑制剂增强了 hRMECs 的细胞生长、迁移和血管生成。此外，上调 miR-199a-3p 可明显减轻 HG 引起的细胞增殖、迁移和血管生成增加。此外，荧光素酶报告基因实验表明，miR-199a-3p 可直接靶向 VEGF。miR-199a-3p 的过表达明显抑制了 HG 刺激的 PI3K/AKT 信号通路和血管生成，LY294002 可进一步抑制该作用。此外，LY294002 可轻微改善 miR-199a-3p 抑制剂刺激的 PI3K/AKT 信号通路和血管生成。
结论：miR-199a-3p 的上调通过抑制 VEGF 调节 PI3K/AKT 通路改善了 HG 刺激的 hRMECs 血管生成。虽然尚未研究体内视网膜新生血管形成，但这些体外发现为 miR-199a-3p 上调对抗 HG 诱导的血管生成提供了更多证据。
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