微小 RNA-15b 通过 Wnt/β-连环蛋白通路调节 IRS-1 参与大鼠糖尿病视网膜病变。
MicroRNA-15b participates in diabetic retinopathy in rats through regulating IRS-1 via Wnt/β-catenin pathway.
机构信息
Department of Ophthalmology, First Affiliated Hospital of Jiamusi University, Jiamusi, China.
出版信息
Eur Rev Med Pharmacol Sci. 2018 Aug;22(16):5063-5070. doi: 10.26355/eurrev_201808_15698.
OBJECTIVE
To explore the role of microRNA-15b in diabetic retinopathy (DR) and its underlying mechanism.
MATERIALS AND METHODS
Diabetic retinopathy rat model was first constructed. Retinal endothelial cells (EC) and retinal pericytes (RP) in DR rats were extracted. The mRNA expression of microRNA-15b in EC and RP cells was detected by qRT-PCR (quantitative Real Time-Polymerase Chain Reaction). Protein expression of insulin receptor substrate 1 (IRS-1) in EC and RP cells was detected by Western blot. After altering microRNA-15b expression by plasmid transfection, cell viability was detected by CCK-8 (cell counting kit-8) assay. Furthermore, the target gene of microRNA-15b was predicted by TargetScan analysis and the binding condition was verified by luciferase reporter gene assay. Finally, rescue experiments were carried out to explore the regulatory effect of microRNA-15b on IRS-1.
RESULTS
MicroRNA-15b was lowly expressed, whereas IRS-1 was highly expressed in EC and RP cells. After overexpression of microRNA-15b, viabilities of EC and RP cells were decreased and β-catenin expression was inhibited. TargetScan predicted that IRS-1 was the downstream gene of microRNA-15b, which was further verified by luciferase reporter gene assay. Rescue experiments indicated that microRNA-15b was capable of regulating IRS-1 via Wnt/β-catenin signaling pathway.
CONCLUSIONS
MicroRNA-15b participates in the development of diabetic retinopathy by targeting IRS-1 via Wnt/β-catenin signaling pathway.
目的
探讨 microRNA-15b 在糖尿病视网膜病变（DR）中的作用及其机制。
材料与方法
首先构建糖尿病视网膜病变大鼠模型，提取 DR 大鼠视网膜内皮细胞（EC）和视网膜周细胞（RP），qRT-PCR 检测 EC 和 RP 细胞中 microRNA-15b 的 mRNA 表达。Western blot 检测 EC 和 RP 细胞中胰岛素受体底物 1（IRS-1）的蛋白表达。通过质粒转染改变 microRNA-15b 的表达，用 CCK-8 法检测细胞活力。此外，通过 TargetScan 分析预测 microRNA-15b 的靶基因，并通过荧光素酶报告基因检测验证其结合情况。最后，进行挽救实验以探讨 microRNA-15b 对 IRS-1 的调节作用。
结果
microRNA-15b 在 EC 和 RP 细胞中低表达，IRS-1 高表达。过表达 microRNA-15b 后，EC 和 RP 细胞活力降低，β-catenin 表达受抑制。TargetScan 预测 IRS-1 是 microRNA-15b 的下游基因，荧光素酶报告基因检测进一步验证了这一点。挽救实验表明，microRNA-15b 可以通过 Wnt/β-catenin 信号通路调节 IRS-1。
结论
microRNA-15b 通过 Wnt/β-catenin 信号通路靶向 IRS-1 参与糖尿病视网膜病变的发生。
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