| 刘 轩,程育宏,齐 赟,崔丽珺,丁国龙,杨 文,陈 丽,谢安明,康前雁.miR-939-5p对糖尿病视性网膜病变和视网膜微血管内皮细胞的调控作用[J].,2021,(22):4249-4255 |
| miR-939-5p对糖尿病视性网膜病变和视网膜微血管内皮细胞的调控作用 |
| The Regulatory Effect of miR-939-5p on Diabetic Retinopathy and Retinal Microvascular Endothelial Cells |
| 投稿时间：2021-03-28 修订日期：2021-04-24 |
| DOI：10.13241/j.cnki.pmb.2021.22.010 |
| 中文关键词: 糖尿病性视网膜病变 miR-939-5p 诱导型一氧化氮合酶 人视网膜微血管内皮细胞 |
| 英文关键词: Diabetic retinopathy MiR-939-5p Inducible nitric oxide synthase Human retinal microvascular endothelial cells |
| 基金项目:国家自然科学基金项目（81800824）；陕西省自然科学基础研究计划项目（2020JM-400）；陕西省自然科学基金项目( 2020SF268) |
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| 中文摘要: |
| 摘要 目的：探究miR-939-5p对糖尿病性视网膜病变和人视网膜微血管内皮细胞（HRMEC）的调控作用。方法：将miR-939-5p模拟物（miR-939-5p-mimic）或miR-939-5p抑制剂（miR-939-5p-inhibitor）转染到HRMEC中，并将细胞用高糖（HG组，25 mM）或低糖（LG组，5 mM）处理24 h。通过细胞计数试剂盒8（CCK-8）来检测细胞活力，EdU法检测细胞DNA的复制能力，Hoechst 33258染色检测细胞凋亡，使用双荧光素酶试剂盒E2920验证miR-939-5p与NOS2 3'-UTR之间的结合关系。对大鼠腹腔注射65 mg/kg的链脲佐菌素（STZ）诱导DR模型，通过RT-qPCR检测miR-939-5p水平，Western Blot检测诱导型一氧化氮合酶（NOS2）水平，苏木精伊红（HE）染色检查大鼠视网膜形态，免疫组织化学染色检测视网膜Claudin-5和Occludin的表达，伊文思蓝染色检测大鼠血视网膜屏障（BRB）通透性，ELISA法检测大鼠房水中白介素-1β（IL-1β）和肿瘤坏死因子-α（TNF-α）的水平。结果：与LG组的HRMEC相比，HG组的miR-939-5p显著降低，而NOS2蛋白水平显著升高（P<0.05）。荧光素酶活性测定显示，与NC-mimic组相比，miR-939-5p-mimic与pGL3-NOS2-WT共转染组的荧光素酶活性显著降低（P<0.05）。与HG+NC-mimic组相比，HG+miR-939-5p-mimic组的miR-939-5p水平和细胞活力显著升高，而NOS2蛋白水平和细胞凋亡率显著降低（P<0.05）。与DR组相比，miR-939-5p-Agomir组大鼠视网膜组织病变减轻，Claudin-5和Occludin的表达水平明显升高，伊文思蓝浓度显著降低（P<0.05）；与DR组相比，miR-939-5p-Agomir组大鼠房水中IL-1β和TNF-α的水平均显著降低（P<0.05）。结论：在高糖培养的HRMEC中和DR大鼠视网膜中，miR-939-5p为低表达模式，NOS2为高表达模式。上调miR-939-5p通过靶向抑制NOS2对DR大鼠视网膜和HRMEC提供保护作用。 |
| 英文摘要: |
| ABSTRACT Objective: To investigate the regulatory effect of miR-939-5p on diabetic retinopathy (DR) and human retinal microvascular endothelial cells (HRMEC). Methods: miR-939-5p-mimic or miR-939-5p-inhibitor was transfected into HRMEC, and the cells were treated with high glucose (HG, 25mM) or low glucose (LG, 5mM) for 24 hours. Cell viability was detected by cell counting kit 8 (CCK-8), DNA replication ability was detected by EdU method, and apoptosis was detected by Hoechst33258 staining. The binding relationship between miR-939-5p and NOS2 3'-UTR was verified by double luciferase kit E2920. The rat model of DR was induced by intraperitoneal injection of streptozotocin (STZ) of 65mg/kg. The level of miR-939-5p was detected by RT-qPCR, the level of inducible nitric oxide synthase (NOS2) was detected by Western Blot, the morphology of rat retina was examined by hematoxylin-eosin (HE) staining, the expression of Claudin-5 and Occludin was detected by immunohistochemical staining, and the permeability of the blood retinal barrier (BRB) in rats was detected by Evans blue staining. The levels of interleukin-1 β(IL-1 β) and tumor necrosis factor-α (TNF-α) in aqueous humor of rats were detected by ELISA. Results: Compared with HRMEC in LG group, miR-939-5p in HG group was significantly reduced, while NOS2 protein level was significantly increased(P<0.05). The luciferase activity measurement showed that, compared with NC-mimic group, the luciferase activity of the miR-939-5p-mimic and pGL3-NOS2-WT co-transfected group was significantly reduced(P<0.05). Compared with HG+NC-mimic group, the miR-939-5p level and cell viability of HG+miR-939-5p-mimic group were significantly increased, while the NOS2 protein level and the apoptosis rate were significantly reduced (P<0.05). Compared with DR group, rats in miR-939-5p-Agomir group had less retinal tissue lesions, the expression levels of Claudin-5 and Occludin were significantly increased, and the concentration of Evans blue was significantly reduced (P<0.05). Compared with DR group, the levels of IL-1β and TNF-α in the aqueous humor of rats in miR-939-5p-Agomir group were significantly reduced (P<0.05). Conclusion: In the high glucose cultured HRMEC and DR rat retina, miR-939-5p is a low expression pattern, and NOS2 is a high expression pattern. Up-regulation of miR-939-5p provides a protective effect on the retina and HRMEC of DR rats by targeting NOS2. |
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