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ENGLISH ABSTRACT
miR-146a对糖尿病视网膜病变炎症反应的抑制作用
顾顺
詹鹏飞
王文娟
王晓露
魏婷婷
朱凌鹏
王杨宁致
殷丽
谢田华
姚勇
作者及单位信息
·
DOI： 10.3760/cma.j.cn115989-20190213-00051
Inhibitory effects of miR-146a on retinal inflammation induced by high glucose in human retinal endothelial cells
Gu Shun
Zhan Pengfei
Wang Wenjuan
Wang Xiaolu
Wei Tingting
Zhu Lingpeng
Wang Yangningzhi
Yin Li
Xie Tianhua
Yao Yong
Authors Info & Affiliations
Gu Shun
Department of Ophthalmology, Wuxi People's Hospital Affiliated to Nanjing Medical University, Wuxi 214023, China
Zhan Pengfei
Department of Ophthalmology, Wuxi People's Hospital Affiliated to Nanjing Medical University, Wuxi 214023, China
Wang Wenjuan
Center of Clinical Research, Wuxi People's Hospital Affiliated to Nanjing Medical University, Wuxi 214023, China
Wang Xiaolu
Center of Clinical Research, Wuxi People's Hospital Affiliated to Nanjing Medical University, Wuxi 214023, China
Wei Tingting
Center of Clinical Research, Wuxi People's Hospital Affiliated to Nanjing Medical University, Wuxi 214023, China
Zhu Lingpeng
Center of Clinical Research, Wuxi People's Hospital Affiliated to Nanjing Medical University, Wuxi 214023, China
Wang Yangningzhi
Department of Ophthalmology, Wuxi People's Hospital Affiliated to Nanjing Medical University, Wuxi 214023, China
Yin Li
Department of Ophthalmology, Wuxi People's Hospital Affiliated to Nanjing Medical University, Wuxi 214023, China
Xie Tianhua
Department of Ophthalmology, Wuxi People's Hospital Affiliated to Nanjing Medical University, Wuxi 214023, China
Yao Yong
Department of Ophthalmology, Wuxi People's Hospital Affiliated to Nanjing Medical University, Wuxi 214023, China
·
DOI： 10.3760/cma.j.cn115989-20190213-00051
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摘要

目的研究微小RNA-146a(miR-146a)对高糖诱导的人视网膜内皮细胞炎症反应的调控作用及其机制。

方法收集2013年10—12月在南京医科大学附属无锡人民医院就诊的单纯糖尿病患者57例和糖尿病视网膜病变(DR)患者40例。另选取41名健康者作为正常对照。收集受检者的一般检查结果，并采集受检者静脉抗凝血各5 ml。体外培养人视网膜内皮细胞(HREC)系，分为高糖组和正常对照组，分别采用含30 mmol/L葡萄糖的DMEM培养液和正常DMEM培养液培养。采用荧光定量PCR法分别检测各组受检者外周血和体外培养HREC中miR-146a的表达水平。分别用lipofectamine2000装载50 nmol/L miR-146a模拟物、模拟物对照剂和miR-146a抑制物、抑制物对照剂转染HREC。采用荧光定量PCR法检测各转染组细胞中miR-146a和细胞间黏附分子-1(ICAM-1)mRNA的表达水平。采用Western blot法检测核因子кB(NF-кB)p65和NF-кB p65 Ser536蛋白相对表达量。

结果糖尿病组和DR组miR-146a相对表达量分别为0.36±0.08和0.27±0.08，明显低于正常对照组的1.00±0.16，差异均有统计学意义(均 P<0.01)。在高糖组HREC细胞中miR-146a相对表达量为0.37±0.11，明显低于正常对照组的1.00±0.18，差异有统计学意义( t＝5.57， P<0.01)。miR-146a模拟物组miR-146a mRNA的相对表达量为2 540.00±105.00，明显高于模拟物对照组的61.00±17.90；miR-146a抑制物组miR-146a mRNA的相对表达量为0.04±0.01，明显低于抑制物对照组的0.88±0.04，差异均有统计学意义( t＝23.23、17.12，均 P<0.01)。miR-146a模拟物组ICAM-1 mRNA的相对表达量为0.35±0.12，明显低于模拟物对照组的1.00±0.13；miR-146a抑制物组ICAM-1 mRNA的相对表达量为2.74±0.48，明显高于抑制物对照组的1.00±0.16，差异均有统计学意义( t＝3.58、3.37，均 P<0.05)。miR-146a模拟物组NF-кB p65 Ser536蛋白相对表达量为0.43±0.03，明显低于模拟物对照组的1.07±0.09，差异有统计学意义( t＝6.74， P<0.01)。miR-146a抑制物组NF-кB p65 Ser536蛋白相对表达量为2.08±0.12，明显高于抑制物对照组的1.00±0.01，差异有统计学意义( t＝8.76， P<0.01)。

结论miR-146a能够通过抑制NF-кB磷酸化水平和ICAM-1的表达，减轻DR的炎症反应。

糖尿病视网膜病变;微小RNA-146a;人视网膜内皮细胞;细胞间黏附分子;核因子κB
ABSTRACT

ObjectiveTo observe the effects of miR-146a on human retinal endothelial cell (HREC) under high glucose condition.

MethodsTotal of 57 cases diagnosed as diabetic mellitus and 40 cases with diabetic retinopathy (DR) in Wuxi People's Hospital Affiliated to Nanjing Medical University from October to December 2013.Forty-one healthy volunteers were enrolled and served as control group.The clinical data and venous blood samples of subjects were collected.HRECs were cultured in normal glucose (5.5 mmol/L) or high glucose medium (30 mmol/L). Real-time PCR was used to detect the expression of miR-146a.The cultured HRECs were transfected with miR-146a mimic, mimic negative control, inhibitor and inhibitor negative control by lipofectamine2000, respectively.The expression of miR-146a and intercellular cell adhesion molecule-1 (ICAM-1) mRNA was examined by real-time PCR and the expression of nuclear factor-кB (NF-кB) p65 and NF-кB p65 Ser536 was detected by Western blot assay.

ResultsThe relative expression of miR-146a mRNA in the diabetic mellitus group and DR group was 0.36±0.08 and 0.27±0.08, respectively, which were significantly lower than 1.00±0.16 in the control group (both at P<0.01). The expression of miR-146a mRNA was 0.37±0.11 in the high glucose group, which was lower than 1.00±0.18 in the normal control group ( t=5.57, P<0.01). The relative expression of miR-146a mRNA in the miR-146a mimic group was 2 540.00±105.00, which was significantly higher than 61.00±17.90 in the miR-146a mimic control group; The relative expression of miR-146a mRNA in the miR-146a inhibitor group was 0.04±0.01, which was significantly lower than 0.88±0.04 in the miR-146a inhibitor control group ( t=23.23, 17.12; both at P<0.01). The relative expression of ICAM-1 mRNA in the miR-146a mimic group was 0.35±0.12, which was significantly lower than 1.00±0.13 in the miR-146a mimic control group; The relative expression of ICAM-1 mRNA in the miR-146a inhibitor group was 2.74±0.48, which was significantly higher than 1.00±0.16 in the miR-146a inhibitor control group ( t=3.58, 3.37; both at P<0.05). The relative expression of NF-кB p65 Ser536 in the miR-146a mimic group was 0.43±0.03, which was significantly lower than 1.07±0.09 in the miR-146a mimic control group ( t=6.74, P<0.01). The relative expression of NF-кB p65 Ser536 in the miR-146a inhibitor group was 2.08±0.12, which was significantly higher than 1.00±0.01 in the miR-146a inhibitor control group ( t=8.76; P<0.01).

ConclusionsmiR-146a can reduce inflammation of HREC in high glucose condition through inhibiting ICAM-1 expression and NF-кB phosphorylation.

Diabetic retinopathy;miR-146a;Human retinal endothelial cell;Intercellular cell adhesion molecule;Nuclear factor kappa B
Yao Yong, Email: mocdef.6ab211draP
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引用本文

顾顺,詹鹏飞,王文娟,等. miR-146a对糖尿病视网膜病变炎症反应的抑制作用[J]. 中华实验眼科杂志,2020,38(09)：733-739.

DOI:10.3760/cma.j.cn115989-20190213-00051

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糖尿病视网膜病变(diabetic retinopathy，DR)是糖尿病患者常见的眼部并发症，是常见致盲眼病，糖尿病患者的高致残率多由糖尿病血管病变所致 [ 1 , 2 ]。视网膜血管是糖尿病早期病理损害的靶器官，高血糖是导致DR的主要危险因素 [ 3 , 4 ]，高血糖可促使视网膜血管渗漏、炎症反应、新生血管和纤维增生膜形成 [ 5 ]。炎性因子是介导糖尿病并发症发病的潜在因素，其通过改变微小RNA(microRNA，miRNA)的表达参与糖尿病患者的表观遗传调控 [ 6 ]。ICAM-1作为一个炎性因子在多种细胞中表达，参与单核细胞和淋巴细胞在内皮细胞的聚集、黏附，其位于19号染色体上，对糖尿病高度易感 [ 7 , 8 ]。之前有研究显示，ICAM-1的表达水平与胰岛素抵抗呈正相关 [ 9 ]。然而DR的具体发病机制以及其新型治疗靶点仍有待进一步探索。miRNA是一类高度保守的非编码小分子RNA，其通过与mRNA完全或不完全配对降解靶mRNA或阻遏其转录后翻译，参与调控细胞发育、分化、生长、凋亡等生理过程 [ 10 , 11 , 12 ]。有研究表明，miRNA可通过靶向调控缺氧诱导因子-1(hypoxia inducible factor-1，HIF-1)/血管内皮生长因子(vascular endothelial growth factor，VEGF)和p53等与DR发生和发展密切相关的靶基因产生不同的细胞生物学效应，从而广泛调节视网膜各类细胞炎症发生、新生血管形成、增生和凋亡等病理过程 [ 13 ]。最近研究表明，miR-146a通过参与免疫炎症反应来调控线粒体功能 [ 14 ]。本团队前期临床样本芯片分析结果显示，糖尿病无DR患者与DR患者外周血中miR-146a有明显差异 [ 15 ]。本研究检测正常对照人群、糖尿病患者以及DR患者血浆中miR-146a表达变化，分析miR-146a在高糖条件下对人视网膜内皮细胞(human retinal endothelial cell，HREC)核因子кB(nuclear factor-кB，NF-κB)通路的影响，并探讨miR-146a在DR中可能的作用机制。
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备注信息
A
姚勇，Email： mocdef.6ab211draP
B
所有作者均声明不存在利益冲突
C
国家自然科学基金项目 (81770941、81800845)
江苏省自然科学基金项目 (BK20180170)
江苏省重点医学学科项目 (ZDXKC2016008)
无锡市卫生健康委员会青年项目 (Q201707)
南京医科大学科技发展基金项目 (2016NJMUZD074、NMUB2018363)
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