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

作　　者：刘轩[1] 程育宏[1] 齐赟 崔丽珺[1] 丁国龙 杨文[3] 陈丽[1] 谢安明[1] 康前雁[1] LIU Xuan;CHENG Yu-hong;QI Yun;CUI Li-jun;DING Guo-long;YANG Wen;CHEN Li;XIE An-ming;KANG Qian-yan(Department of Ophthalmology,the First Affiliated Hospital of Xi'an Jiaotong University,Xi'an,Shaanxi,710061,China;Department of Ophthalmology,Xian No.1 Hospital,Xi'an,Shaanxi,710002,China;Department of Ophthalmology,Xian Fourth Hospital,Xi'an,Shaanxi,710004,China)

机构地区：[1]西安交通大学第一附属医院眼科,陕西西安710061 [2]西安市第一医院眼科,陕西西安710002 [3]西安市第四医院眼科,陕西西安710004

出　　处：《现代生物医学进展》2021年第22期4249-4255,4307,共8页Progress in Modern Biomedicine

基　　金：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)。

摘　　要：目的:探究mi R-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mi R-939-5p模拟物(mi R-939-5p-mimic)或mi R-939-5p抑制剂(mi R-939-5p-inhibitor)转染到HRMEC中,并将细胞用高糖(HG组,25 m M)或低糖(LG组,5 m M)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mi R-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mi R-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mi R-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,mi R-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+mi R-939-5p-mimic组的mi R-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,mi R-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,mi R-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,mi R-939-5p为低表达模式,NOS2为高表达模式。上调mi R-939-5p通过靶向抑制NOS2对DR大鼠视网膜和HRMEC提供保护作用。Objective:To investigate the regulatory effect of mi R-939-5 p on diabetic retinopathy(DR)and human retinal microvascular endothelial cells(HRMEC).Methods:mi R-939-5 p-mimic or mi R-939-5 p-inhibitor was transfected into HRMEC,and the cells were treated with high glucose(HG,25 mM)or low glucose(LG,5 mM)for 24 hours.Cell viability was detected by cell counting kit8(CCK-8),DNA replication ability was detected by EdU method,and apoptosis was detected by Hoechst33258 staining.The binding relationship between mi R-939-5 p and NOS23’-UTR was verified by double luciferase kit E2920.The rat model of DR was induced by intraperitoneal injection of streptozotocin(STZ)of 65 mg/kg.The level of mi R-939-5 p 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-1β(IL-1β)and tumor necrosis factor-α(TNF-α)in aqueous humor of rats were detected by ELISA.Results:Compared with HRMEC in LG group,mi R-939-5 p 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 mi R-939-5 p-mimic and pGL3-NOS2-WT co-transfected group was significantly reduced(P<0.05).Compared with HG+NC-mimic group,the mi R-939-5 p level and cell viability of HG+mi R-939-5 p-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 mi R-939-5 p-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-1βand TNF-�

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 

分 类 号：R-33[医药卫生] R774.1