微小 RNA-15b 的低表达通过上调糖尿病大鼠中 VEGFA 促进视网膜毛细血管内皮细胞和周细胞的增殖。
Low expression of microRNA-15b promotes the proliferation of retinal capillary endothelial cells and pericytes by up-regulating VEGFA in diabetic rats.
机构信息
Department of Ophthalmology, Binzhou City Center Hospital, Binzhou, China.
出版信息
Eur Rev Med Pharmacol Sci. 2019 Jul;23(14):6018-6025. doi: 10.26355/eurrev_201907_18413.
OBJECTIVE
To investigate the role of microRNA-15b in diabetic retinopathy and its underlying mechanism.
MATERIALS AND METHODS
Diabetes rat model was established by streptozotocin injection. The mRNA expression of microRNA-15b in retinal capillary endothelial cells and pericytes of diabetic rats was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The mRNA and protein expressions of vascular endothelial growth factor A (VEGFA) were detected by qRT-PCR and Western blot, respectively. MicroRNA-15b mimics or inhibitor were transfected into retinal capillary endothelial cells and pericytes of diabetic rats, respectively. The mRNA expressions of microRNA-15b and VEGFA were detected by qRT-PCR. Cell counting kit-8 (CCK-8) assay was used to detect the proliferation of capillary endothelial cells and pericytes. Dual-Luciferase reporter gene assay was conducted to verify the binding condition of microRNA-15b and VEGFA. RNA immunoprecipitation (RIP) assay was performed to determine whether microRNA-15b could bind to AGO2. Rescue experiments were finally carried out by detecting the proliferation of retinal capillary endothelial cells and pericytes after downregulation or overexpression of microRNA-15b and VEGFA.
RESULTS
The expression of microRNA-15b decreased, whereas VEGFA expression increased in retinal capillary endothelial cells and pericytes of diabetic rats. High expression of microRNA-15b in retinal capillary endothelial cells and pericytes resulted in VEGFA down-regulation and decreased proliferation. RIP assay results indicated that microRNA-15b could interact with AGO2. Additionally, Dual-Luciferase reporter gene assay showed that VEGFA is a direct target gene of microRNA-15b. VEGFA overexpression could reverse the inhibited proliferation of retinal capillary endothelial cells and pericytes induced by microRNA-15b overexpression. Similarly, VEGFA knockdown could reverse the effect of the low expression of microRNA-15b on the proliferation of retinal capillary endothelial cells and pericytes.
CONCLUSIONS
Low expression of microRNA-15b in retinal capillary endothelial cells and pericytes of diabetic rats promotes the development of diabetic retinopathy by up-regulating VEGFA.
目的
探讨 microRNA-15b 在糖尿病视网膜病变中的作用及其机制。
材料和方法
采用链脲佐菌素注射建立糖尿病大鼠模型。采用实时定量聚合酶链反应（qRT-PCR）检测糖尿病大鼠视网膜毛细血管内皮细胞和周细胞中 microRNA-15b 的 mRNA 表达。采用 qRT-PCR 和 Western blot 分别检测血管内皮生长因子 A（VEGFA）的 mRNA 和蛋白表达。分别将 microRNA-15b 模拟物或抑制剂转染至糖尿病大鼠视网膜毛细血管内皮细胞和周细胞，采用 qRT-PCR 检测 microRNA-15b 和 VEGFA 的 mRNA 表达。采用细胞计数试剂盒-8（CCK-8）检测毛细血管内皮细胞和周细胞的增殖。采用双荧光素酶报告基因实验验证 microRNA-15b 与 VEGFA 的结合情况。采用 RNA 免疫沉淀（RIP）实验确定 microRNA-15b 是否能与 AGO2 结合。最后通过下调或过表达 microRNA-15b 和 VEGFA 检测视网膜毛细血管内皮细胞和周细胞的增殖，进行挽救实验。
结果
糖尿病大鼠视网膜毛细血管内皮细胞和周细胞中 microRNA-15b 表达降低，而 VEGFA 表达升高。视网膜毛细血管内皮细胞和周细胞中高表达 microRNA-15b 导致 VEGFA 下调和增殖减少。RIP 实验结果表明，microRNA-15b 可与 AGO2 相互作用。此外，双荧光素酶报告基因实验表明，VEGFA 是 microRNA-15b 的直接靶基因。VEGFA 过表达可逆转 microRNA-15b 过表达抑制的视网膜毛细血管内皮细胞和周细胞增殖。同样，下调 VEGFA 可逆转 microRNA-15b 低表达对视网膜毛细血管内皮细胞和周细胞增殖的影响。
结论
糖尿病大鼠视网膜毛细血管内皮细胞和周细胞中 microRNA-15b 的低表达通过上调 VEGFA 促进糖尿病视网膜病变的发展。
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