[1]王艳平,刘含军.长链非编码RNA（LncRNA）母系表达基因3（MEG3）对糖尿病视网膜病变大鼠模型视网膜血管内皮细胞凋亡的影响[J].眼科新进展,2023,43(1):018-24.[doi:10.13389/j.cnki.rao.2023.0004]
　WANG Yanping,LIU Hanjun.Effect of long non-coding ribonucleic acid maternally expressed gene 3 on retinal vascular endothelial cell apoptosis in rats with diabetes retinopathy[J].Recent Advances in Ophthalmology,2023,43(1):018-24.[doi:10.13389/j.cnki.rao.2023.0004]
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长链非编码RNA（LncRNA）母系表达基因3（MEG3）对糖尿病视网膜病变大鼠模型视网膜血管内皮细胞凋亡的影响/HTML
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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
43卷
期数:
2023年1期
页码:
018-24
栏目:
实验研究
出版日期:
2023-01-05

文章信息/Info

Title:
Effect of long non-coding ribonucleic acid maternally expressed gene 3 on retinal vascular endothelial cell apoptosis in rats with diabetes retinopathy
作者:
王艳平; 刘含军
433000　湖北省仙桃市，长江大学附属仙桃市第一人民医院眼科
Author(s):
WANG Yanping; LIU Hanjun
Department of Ophthalmology,Xiantao First People’s Hospital Affiliated to Changjiang University,Xiantao 433000,Hubei Province,China
关键词:
长链非编码RNA; 母系表达基因3; miR-145-5p; 糖尿病视网膜病变; 视网膜血管内皮细胞凋亡
Keywords:
long non-coding ribonucleic acid;  maternally expressed gene 3;  miR-145-5p;  diabetic retinopathy;  retinal vascular endothelial cell apoptosis
分类号:
R774
DOI:
10.13389/j.cnki.rao.2023.0004
文献标志码:
A
摘要:
目的　探讨长链非编码RNA（LncRNA）母系表达基因3（MEG3）对糖尿病视网膜病变（DR）大鼠模型视网膜血管内皮细胞凋亡的影响。
方法　取SPF级雄性SD大鼠以及体外培养的人视网膜微血管内皮细胞（HRMEC），均随机分为对照组、模型组、LncRNA MEG3过表达组、miR-145-5p agomir组、共转染（LncRNA MEG3过表达质粒+miR-145-5p agomir）组、共转染阴性对照（LncRNA MEG3空载质粒+miR-145-5p agomir阴性对照质粒）组，采用腹腔注射链脲佐菌素建立DR大鼠模型，25 mmol·L-1葡萄糖诱导DR细胞模型。对照组大鼠腹腔注射柠檬酸缓冲液，对照组细胞以正常培养基培养，将大鼠与细胞均分组进行处理，以LncRNA MEG3质粒、miR-145-5p agomir转染处理后，通过伊文思蓝（EB）检测大鼠视网膜血管通透性；TUNEL染色检测大鼠视网膜血管内皮细胞凋亡率；EdU染色与Hoechst 33258染色分别检测细胞增殖、凋亡情况；依照试剂盒测量大鼠房水中及HRMEC炎症因子白细胞介素（IL）-6、IL-1β含量；实时荧光定量PCR实验检测HRMEC及大鼠视网膜组织miR-145-5p表达；蛋白免疫印迹法检测HRMEC及大鼠视网膜组织凋亡相关蛋白（Bcl-2、Bax）表达；双荧光素酶实验验证HRMEC中LncRNA MEG3对miR-145-5p的靶向调节作用。
结果　LncRNA MEG3过表达组大鼠玻璃体中EB含量、视网膜血管内皮细胞凋亡率及房水中IL-6、IL-1β含量均低于模型组、共转染组，差异均有统计学意义（均为P<0.05）；miR-145-5p agomir组大鼠玻璃体中EB含量、视网膜血管内皮细胞凋亡率及房水中IL-6、IL-1β含量均高于模型组、共转染组，差异均有统计学意义（均为P<0.05）。LncRNA MEG3过表达组HRMEC增殖率均高于模型组、共转染组，差异均有统计学意义（均为P<0.05），HRMEC凋亡率及IL-6、IL-1β含量均低于模型组、共转染组，差异均有统计学意义（均为P<0.05）；miR-145-5p agomir组HRMEC增殖率均低于模型组、共转染组，差异均有统计学意义（均为P<0.05），HRMEC凋亡率及IL-6、IL-1β含量均高于模型组、共转染组，差异均有统计学意义（均为P<0.05）。LncRNA MEG3过表达组HRMEC、大鼠视网膜组织miR-145-5p及Bax表达均低于模型组、共转染组，Bcl-2表达均高于模型组、共转染组，差异均有统计学意义（均为P<0.05）；miR-145-5p agomir组HRMEC、大鼠视网膜组织miR-145-5p及Bax表达均高于模型组、共转染组，Bcl-2表达均低于模型组、共转染组，差异均有统计学意义（均为P<0.05）。转染野生型miR-145-5p报告质粒+LncRNA MEG3过表达质粒组细胞相对荧光素酶活性（0.33±0.05）低于转染野生型miR-145-5p报告质粒+LncRNA MEG3空载质粒组（1.02±0.21），差异有统计学意义（P<0.05）。
结论　LncRNA MEG3可通过靶向下调miR-145-5p表达，抑制炎症反应，减轻DR大鼠视网膜血管内皮细胞凋亡，促使HRMEC DR细胞模型存活，改善DR大鼠视网膜血管屏障功能。
Abstract:
Objective　To investigate the effect of long non-coding ribonucleic acid (LncRNA) maternally expressed gene 3 (MEG3) on apoptosis of retinal vascular endothelial cells in diabetes retinopathy (DR) rats.
Methods　Specific pathogen-free male Sprague-Dawley rats and human retinal microvascular endothelial cells (HRMEC) cultured in vitro were randomly divided into control group, model group, LncRNA MEG3 overexpression group, miR-145-5p agomir group, co-transfection (LncRNA MEG3 overexpression plasmid + miR-145-5p agomir) group, and co-transfection negative control (LncRNA MEG3 empty plasmid + miR-145-5p agomir negative control plasmid) group. DR rat model was established by intraperitoneal injection of streptozotocin, and the DR cell model was induced by 25 mmol·L-1 glucose. The rats in the control group were intraperitoneally injected with citrate acid buffer, and the cells in the control group were cultured in a normal medium. The rats and cells were divided into groups and transfected with LncRNA MEG3 plasmid and miR-145-5p agomir, then the retinal vascular permeability was detected by Evans blue (EB); the apoptosis rate of retinal vascular endothelial cells was detected by TUNEL staining; cell proliferation and apoptosis were detected by EdU and Hoechst 33258 staining, respectively; the contents of inflammatory factor interleukin (IL)-6 and IL-1β in the aqueous humor of rats and HRMEC were measured with kits; the expression of miR-145-5p in HRMEC and rat retinal tissue was detected by real-time quantitative PCR experiment; the expressions of apoptosis-related proteins (Bcl-2 and Bax) in HRMEC and rat retina were detected by western blot; the targeted regulation of LncRNA MEG3 on miR-145-5p in HRMEC was verified by dual-luciferase experiments.
Results　The content of EB in the vitreous body, the apoptosis rate of retinal vascular endothelial cells, and the contents of IL-6 and IL-1β in the aqueous humor of rats in the LncRNA MEG3 overexpression group were lower than those in the model group and co-transfection group, and the differences were statistically significant (all P<0.05); the content of EB in vitreous body, the apoptosis rate of retinal vascular endothelial cells, the contents of IL-6 and IL-1β in aqueous humor of rats in the miR-145-5p agomir group were higher than those in the model group and co-transfection group, and the differences were statistically significant (all P<0.05). The proliferation rate of HRMEC in the LncRNA MEG3 overexpression group was significantly higher than that in the model group and co-transfection group (both P<0.05), and the apoptosis rate of HRMEC and the contents of IL-6, IL-1β were significantly lower than those in the model group and co-transfection group (all P<0.05); the proliferation rate of HRMEC in the miR-145-5p agomir group was significantly lower than that in the model group and co-transfection group (both P<0.05), and the apoptosis rate of HRMEC and the contents of IL-6, IL-1β were significantly higher than those in the model group and co-transfection group (all P<0.05). The expressions of miR-145-5p and Bax in HRMEC and rat retinal tissue in the LncRNA MEG3 overexpression group were lower than those in the model group and co-transfection group, and the expression of Bcl-2 was higher than that in the model group and co-transfection group, with statistical significance (all P<0.05); the expressions of miR-145-5p and Bax in HRMEC and rat retinal tissue in the miR-145-5p agomir group were higher than those in the model group and co-transfection group
Conclusion　LncRNA MEG3 can realize the targeted down-regulation of miR-145-5p expression to inhibit the inflammatory response, reduce the apoptosis of retinal vascular endothelial cells in DR rats, promote the survival of HRMEC DR cell model, and improve retinal vascular barrier function in DR rats.

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备注/Memo

备注/Memo:
长江大学 2019 年度医学科研资助项目（编号：WJ2019-62）
更新日期/Last Update: 2023-01-05