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  

Alterations of microRNA expression profile and bioinformatics analysis in human microvascular reti. nal endothelial cell injured by oxidative stress

作　　者：韩雪 张睿[2] 姜磊[2] 谢冰[2] 崔冬生[2] 许顺江[1] 周慧敏[1] Han Xue , Zhang Rui, Jiang Lei, Xie Bing, Cui Dong- sheng, Xu Shunjiang , Zhou Huimin.(Department of Endocrinology, The First Hospital of Hebei Medical Uni- versity, Shifiazhuang 050031, Chin)

机构地区：[1]河北医科大学第一医院内分泌科,石家庄050031 [2]河北医科大学第一医院中心实验室,石家庄050031

出　　处：《国际内分泌代谢杂志》2018年第2期78-83,共6页International Journal of Endocrinology and Metabolism

基　　金：国家自然科学基金（81570728）

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一8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，100umol／LH2O2干预组8h和16hHUVEC存活率未发生明显改变（F100umol／L=3．897，P〉0．05），其他浓度H2O2干预组细胞存活率明显下降（F200umol/L=8．172、F800umol/L=239．214，P均〈0．05），且呈剂量和时间依赖性。400umol／L干预组24h细胞存活率下降接近50％（F400umol/L=6．905，P〈0．05）。微阵列分析显示，H2O2（400umol／L）孵育HUVEC24h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400umol／L H2O2处理24h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-106h．HUVEC=8．4、FmiRNA497,HUVEC=63．5、FmiRNA-15bHMRFc=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、Fm。RNA-6388．HUVEC=812、FmiRNA-1246．HUVEC=58．5、FmiRNA4267．HUVEE=1179．1、FnuRNA却34，HuvEc=173、FmiRNA-195,himREc=67．8、FmiHMREC=103．7、FmiRNA-1246,himREc=2078．9、FmIRNA-4267HMREc：234．6、FmiRNA4734,HMREC=10．7，P均〈0．05）。结论氧化应激可导致HMREC�Objective To observe the changes of microRNA （miRNA） expression profile during ox- idative 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 ceil （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 bioin- formatics 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 HUYEC detected by CCK-8 assay, the cell viability of HUVEC treated by 100 Ixmol/L H202 for 8 hours and 16 hours were not different from those of the control group （ F^0o^moVL = 3. 897, P 〉 0.05 ）. However, the cell viability of HUVEC treated by other concentrations of H2 02 were decreased significantly and in a dose and time-depend- ent manner（ F200~.mol/L = 8. 172 ,Fs0%moVL = 239. 214, all P 〈 0.05 ）. The cell viability of HUVEC treated with 400 p.mol/L of H202 for 24 hours were decreased by nearly 50% （ F40o^moVL = 6. 905, P 〈 O. 05 ）. The mi- croarray analysis results showed that there were 116 differentially expressed miRNAs in HUVEC after expo- sure to H：O2 （400 p^moL/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 multi- ple biological pathways such as metabolic pathways in cells, adenosine monophosphate activated protein ki- nase（AMPK） signaling pathway and so on. The results of real-time PCR validated that miRNA-15b, miR- NA-106b, and miRNA-497 were significantly downregulated （ FmiRNA1I5b,HUVEC = 9.9, FmIRNA_I06b,I_IUVEC =81. 4, F,niRNA-97,HUVEC - 63. 5,

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 

分 类 号：R587.2[医药卫生—内分泌] R774.1[医药卫生—内科学]