高糖诱导内皮细胞损伤microRNA表达紊乱的体外研究

目的:研究高糖诱导的内皮细胞损伤微小RNA(microRNA,miRNA)的表达变化。方法:常规培养的人冠状动脉内皮细胞,利用不同浓度D-葡萄糖溶液(0 mmol/L、5 mmol/L、15 mmol/L和25 mmol/L),诱导刺激24 h后分别用CCK-8法和流式细胞术检测其生长活力和凋亡水平。收集细胞总RNA,利用实时定量PCR(Quantitative real-time PCR,q RT-PCR)检测miRNA的表达变化,同时利用TargetScan、PicTar等生物信息学预测软件预测可能的靶基因。结果:高糖溶液(25 mmol/L)刺激内皮细胞后,细胞生长活力明显降低,为对照组的67.5%(P0.01),凋亡水平为对照组的4.5倍(P0.01)。QRT-PCR结果显示miRNA的表达出现了明显的紊乱,其中miR-451、miR-504、miR-302d、miR-18b*、miR-198、miR-328和miR-517c明显下调,miR-29c、miR-100*、miR-137、miR-660和miR-217明显上调(P0.05)。靶基因预测发现miR-217和miR-451可能调控内皮细胞功能相关的多个基因的表达。结论:在高糖诱导的内皮细胞损伤中,miRNA表达紊乱提示其可能参与内皮细胞功能。

In Vitro study of MicroRNA Change in High-glucose Induced Endothelial Cell Injury

Objective:To determine the expression change of microRNA (miRNA) in endothelial cell injury induced by high-glucose.Methods:Human coronary artery endothelial cells cultured by conventional methods, were stimulated by using different concentrations of D-glucose solution (0 mmol/L, 5 mmol/L, 15 mmol/L and 25 mmol/L). Cells vitality and apoptosis were measured by CCK-8 assay and flow cytometry. Total RNA was isolated from cells in 24h after high-glucose induced, and miRNA expression levels were detected by using quantitative real-time PCR. Bioinformatics prediction software including Targetscan and PicTar etc, were applied to predict potential target genes of these miRNAs.Results:Endothelial cell viability decreased significantly after high-glucose (25 mmol/L) stimulation, with 67.5%to the control cells (P<0.01), and the apoptosis level were 4.5 times compared with control cells (P<0.01). Quantitative real-time PCR results showed that the expression of miRNA appeared obvious disorder, which were miR-451, miR-504, miR-302d, miR-18b*, miR-198, miR-328 and miR-517c were increased, while miR-29c, miR-100*, miR-137, miR-660 and miR-217 were decreased notably (P<0.05). MiR-217 and miR-451 might regulate the expression of multiple genes related to endothelial cell function by Bioinformatics prediction.Conclusion:The abnormal expression miRNAs in endothelial cells after high glucose induced injury suggested that these miRNAs maybe be involved in regulation of endothelial cells function.