微小RNA芯片检测高糖环境下人视网膜微血管内皮细胞微小RNA的差异表达

Study on the differentially expressed microRNA of human retinal microvascular endothelial cells in high glucose environment by microRNA gene chip

目的筛选高糖环境下人视网膜微血管内皮细胞（HRCEC）差异表达的微小RNA（miRNA），并预测部分差异表达的miRNA调控的靶基因。方法实验研究。常规培养HRCEC，取生长良好的第3～4代HRCEC，将细胞分为3组：（1）正常对照组：培养液为DMEM高糖培养液，含25mmoVL葡萄糖；（2）高糖组：培养液为条件培养液，含90mmol/L葡萄糖；（3）甘露醇高渗对照组：培养液为条件培养液，含65mmoVL甘露醇＋25mmoVL葡萄糖。各组细胞在上述条件下培养5d后，用细胞凋亡检测试剂盒对细胞进行凋亡检测；提取细胞的总RNA并进行质量检测；miRNA芯片检测差异表达的miRNA，并用实时荧光定量PCR对部分差异表达的miRNA进行验证，运用生物信息学方法预测它们可能调控的靶基因。结果在荧光显微镜下观察细胞凋亡结果显示，正常对照组及甘露醇高渗对照组细胞的细胞核被4′，6-二脒基-2-苯基吲哚染色而呈蓝色荧光，但无凋亡荧光信号；高糖组细胞的细胞核也呈蓝色荧光，但有绿色的凋亡荧光信号。总RNA的质量检测结果显示，用分光光度计测量正常对照组细胞吸光度值（A）的比值，A260／A280=1．99、A260／A230=2．05；高糖组细胞A260／A280=1．98、A260／A230=2．26。甲醛变性琼脂糖凝胶电泳见电泳条带清晰完整，表明获得的总RNA质量较好且纯度高。miRNA芯片检测结果显示，与正常对照组相比，在高糖组一共筛选出49个差异表达的miRNAs（上调〉2倍或下调〈0．5倍），其中表达上调的miRNAs31个，表达下调的miRNAs18个。对miRNA芯片检测结果显示在正常对照组和高糖组间表达有差异的has—miR-320c和has—miR-9a^＊进行实时荧光定量PCR验证，结果与芯片结果一致；对这两个miRNAs进行靶基因预测，结果显示这两个基因涉及很多生长因子和蛋白。结论部分miRNA在高糖刺激下的HRCEC中存在差异性表达。

Objectives To explore the differentially expressed microRNA （miRNA） of human retinal rnicrovascular endothelial cell （ HRCEC ） in hyperglycemic environment by miRNA gene chip, then adopt bioinformatics methods to forecast target genes of part differentially expressed miRNA. Methods Experimental study. HRCEC were cultured. Took the 3 -4 generation growth good cells and divided the cells into three groups ： （ 1 ） normal control group： DMEM medium with 25 mmol/L glucose ; （ 2 ） high glucose group： conditioned medium with 90 mmol/L glucose; （3） mannitol high permeability control group： conditioned medium with 65 mmol/L mannitol and 25 mmol/L glucose. Each group ceils were cultured in the above conditions for five days, then used in situ cell death detection kit for apoptosis detection ; the total RNA was isolated and examined; the differentially expressed miRNA were detected by miRNA gene chip, part results of miRNA array were verified by real-time quantitate polymerase chain reaction （ PCR）, potential miRNA targets were analyzed by bioinformatics methods. Results Observed apoptotic HRCEC by fluorescence microscope ： the nucleus of normal control group and mannitol control group were dyed by DAPI and appeared blue fluorescence,but hadn＇t apoptosis fluorescent signals; the nucleus of high glucose groupalso appeared blue fluorescence, and had green apoptosis fluorescent signals. Quality testing of total RNA： with spectrophotometer measurement, the ratio of absorbance of total RNA in normal control group at A260/ A280nm was 1.99, at A260/A230 was 2. 05 ; total RNA of high glucose group at A260/A280was 1.98, at A260/A230was 2. 26. The results of formaldehyde degeneration agarose gel electrophoresis showed that the electrophoresis strips were clear and complete, indicated that the total RNA had better quality and high purity. Compared with normal control group, 49 miRNAs were found to be differentially expressed in high glucose group（ fold change 〉 2 and fold change 〈 0. 5 ） , including 31 up-regulated miRNAs and 18 down- regulated miRNAs. The results of real-time quantitatie PCR revealed that hsa-miR-320c and hsa-miR-29a ^·were up-regulated in high glucose group,which were consistence with the miRNA gene chip. Furthermore, the target genes predction of two above miRNAs were involved many growth factors and proteins. Conclusion miRNA are differently expressed in HRCEC under hyperglycemic conditions.