[1]韩雪 张睿 姜磊 谢冰 崔冬生 许顺江 周慧敏.氧化应激致人视网膜血管内皮细胞损伤中 microRNA表达谱的改变及生物信息学分析[J].国际内分泌代谢杂志,2018,38(02):78-83.[doi:10.3760/cma.j.issn.1673-4157.2018.02.002]
　Han Xue*,Zhang Rui,Jiang Lei,et al.Alterations of microRNA expression profile and bioinformatics analysis in human microvascular retinal endothelial cell injured by oxidative stress[J].International Journal of Endocrinology and Metabolism,2018,38(02):78-83.[doi:10.3760/cma.j.issn.1673-4157.2018.02.002]
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氧化应激致人视网膜血管内皮细胞损伤中 microRNA表达谱的改变及生物信息学分析()
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《国际内分泌代谢杂志》[ISSN:1673-4157/CN:12-1383/R]

卷:
38
期数:
2018年02期
页码:
78-83
栏目:
论著
出版日期:
2018-03-20

文章信息/Info

Title:
Alterations of microRNA expression profile and bioinformatics analysis in human microvascular retinal endothelial cell injured by oxidative stress
作者:
韩雪 张睿 姜磊 谢冰 崔冬生 许顺江 周慧敏
050031 石家庄,河北医科大学第一医院内分泌科(韩雪、周慧敏),中心实验室(张睿、姜磊、谢冰、崔冬生、许顺江)
Author(s):
Han Xue*;  Zhang Rui;  Jiang Lei;  Xie Bing;  Cui Dongsheng;  Xu Shunjiang;  Zhou Huimin.
*Department of Endocrinology, The First Hospital of Hebei Medical University, Shijiazhuang 050031, China
关键词:
氧化应激;  microRNA;  人脐静脉内皮细胞;  人视网膜血管内皮细胞;  生物信息学分析
Keywords:
Oxidative stress;  microRNA;  Human umbilical vein endothelial cells;  Human microvascular retinal endothelial cells;  Bioinformatics analysis
DOI:
10.3760/cma.j.issn.1673-4157.2018.02.002
摘要:
目的 检测氧化应激引起人视网膜血管内皮细胞(HMREC)损伤过程中微小RNA(miRNA)表达谱的改变,并探讨这些差异性表达的miRNA在视网膜血管内皮损伤过程中的作用。方法 利用不同浓度的H2O2溶液孵育人脐静脉内皮细胞(HUVEC)建立氧化应激模型,并用CCK-8细胞增殖实验检测细胞活性。采用基因芯片技术检测氧化应激介导的HUVEC中miRNA表达谱的变化,对差异性表达的miRNA进行生物信息学分析,用实时PCR方法在HMREC中进行验证。结果 CCK-8检测HUVEC活力的结果显示,与同一时间对照组相比,100 μmol/L H2O2干预组8 h和16 h HUVEC存活率未发生明显改变(F100μmol/L=3.897,P>0.05),其他浓度H2O2干预组细胞存活率明显下降(F200μmol/L=8.172、F800μmol/L=239.214,P均<0.05),且呈剂量和时间依赖性。400 μmol/L干预组24 h细胞存活率下降接近50%(F400μmol/L=6.905,P<0.05)。微阵列分析显示,H2O2(400 μmol/L)孵育HUVEC 24 h后,共有116个miRNA的表达发生改变。其中有11个miRNA表达差异在1.5倍以上。生物信息学分析提示,差异表达的miRNA可能参与细胞的代谢调节、AMP活化蛋白激酶(AMPK)信号通路等多个生物学过程。实时PCR检测证实,在经过400 μmol/L H2O2处理24 h后的HUVEC和HMREC的氧化应激模型中,miRNA-15b、miRNA-106b、miRNA-497均显著下调(FmiRNA-15b,HUVEC=9.9、FmiRNA-106b,HUVEC=8.4、FmiRNA-497,HUVEC=63.5、FmiRNA-15b,HMREC=643.7、FmiRNA-106b,HMREC=81.4、FmiRNA-497,HMREC=199.9,P均<0.05),而miRNA-195、miRNA-638、miRNA-1246、miRNA-4267和miRNA-4734均显著上调(FmiRNA-195,HUVEC=592.1、FmiRNA-638,HUVEC=812、FmiRNA-1246,HUVEC=58.5、FmiRNA-4267,HUVEC=1 179.1、FmiRNA-4734,HUVEC=173、FmiRNA-195,HMREC=67.8、FmiRNA-638,HMREC =103.7、FmiRNA-1246,HMREC=2 078.9、FmiRNA-4267,HMREC=234.6、FmiRNA-4734,HMREC=10.7,P均<0.05)。结论 氧化应激可导致HMREC中多个miRNA表达失衡,这些差异性表达的miRNA可能通过AMPK信号通路,在视网膜血管内皮损伤过程中发挥潜在作用。
Abstract:
Objective To observe the changes of microRNA(miRNA)expression profile during oxidative stress in human microvascular retinal endothelial cell(HMREC)and to explore the function of the differentially expressed miRNA in the process of retinal vascular endothelial injury.Methods Oxidative stress model was developed with different concentrations of H2O2 in human umbilical vein endothelial cell(HUVEC). Cell viability was determined by using Cell Counting Kit-8 assay. Gene chip technology was used to detect the alterations of miRNA expression profile induced by oxidative stress in HUVEC and bioinformatics analysis was performed in differentially expressed miRNA. Real-time PCR analysis was used to validate the changed miRNA in HMREC.Results According to the results of viability of HUVEC detected by CCK-8 assay, the cell viability of HUVEC treated by 100 μmol/L H2O2 for 8 hours and 16 hours were not different from those of the control group(F100μmol/L=3.897, P>0.05). However, the cell viability of HUVEC treated by other concentrations of H2O2 were decreased significantly and in a dose and time-dependent manner(F200μmol/L=8.172,F800μmol/L=239.214,all P<0.05). The cell viability of HUVEC treated with 400 μmol/L of H2O2 for 24 hours were decreased by nearly 50%(F400μmol/L=6.905,P<0.05). The microarray analysis results showed that there were 116 differentially expressed miRNAs in HUVEC after exposure to H2O2(400 μmol/L)for 24 hours. Among them, 11 miRNAs were decreased or increased for more than 1.5 times. The results of bioinformatics analysis showed that these miRNAs might be involved in multiple biological pathways such as metabolic pathways in cells, adenosine monophosphate activated protein kinase(AMPK)signaling pathway and so on. The results of real-time PCR validated that miRNA-15b, miRNA-106b, and miRNA-497 were significantly downregulated(FmiRNA-15b,HUVEC=9.9,FmiRNA-106b,HUVEC=8.4,FmiRNA-497,HUVEC=63.5,FmiRNA-15b,HMREC=643.7,FmiRNA-106b,HMREC=81.4,FmiRNA-497,HMREC=199.9,all P<0.05),whereas miRNA-195, miRNA-638, miRNA-1246, miRNA-4267, miRNA-4324, and miRNA-4734 were significantly upregulated(FmiRNA-195,HUVEC=592.1,FmiRNA-638,HUVEC=812,FmiRNA-1246,HUVEC=58.5,FmiRNA-4267,HUVEC=1 179.1,FmiRNA-4734,HUVEC=173,FmiRNA-195,HMREC=67.8,FmiRNA-638,HMREC=103.7,FmiRNA-1246,HMREC=2 078.9,FmiRNA-4267,HMREC=234.6,FmiRNA-4734,HMREC=10.7,all P<0.05)in HUVEC and HMREC oxidative stress model treated by 400 μmol/L of H2O2 for 24 hours.Conclusion Oxidative stress induces the imbalance of miRNA expression in HMREC, and differentially expressed miRNA might play a potential role in the process of retinal vascular endothelial injury through AMPK signaling pathway.

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备注/Memo

备注/Memo:
基金项目:国家自然科学基金(81570728)
通信作者:周慧敏,Email:zhouhuimindoctor@163.com; 许顺江,Email:sjxu66@sina.com
更新日期/Last Update: 1900-01-01