氧化低密度脂蛋白诱导人脐静脉内皮细胞凋亡中microRNA表达谱分析

摘要

目的:研究氧化低密度脂蛋白(ox-LDL)诱导人脐静脉内皮细胞(HUVECs)凋亡过程中micro-RNA(miRNA)表达谱的变化,对差异miRNA 调控的靶基因进行初步预测.方法:建立体外ox-LDL 诱导HUVECs 凋亡模型,采用miRNA 芯片技术筛选表达变化显著的miRNA,实时荧光定量PCR 验证结果,并对差异miRNA 调控的靶基因进行生物信息学分析.结果:miRNA 芯片检测发现,在ox-LDL 诱导HUVECs 凋亡过程中有4 个表达上调和11 个表达下调的miRNA.实时荧光定量PCR 对其中2 个上调(hsa-miR-142-3p 、hsa 蛳miR-365)和2 个下调(hsa-miR-590-5p、hsa-miR-33a)miRNA 的验证结果与芯片检测所示有较好的一致性.生物信息学分析发现,差异miRNA 调控的靶基因与细胞增殖、凋亡、代谢及原癌基因表达等生物学功能相关.结论:经ox-LDL 诱导凋亡的HUVECs 其miRNA 表达谱发生明显改变,提示miRNA 可能参与内皮细胞功能障碍和动脉粥样硬化的发生.%Objective: To observe the expression of microRNA (miRNA) in human umbilical vein endothelial cells (HUVECs) induced by oxidized low-density lipoprotein (ox-LDL) and to make an initial prediction of target genes regulated by differentially expressed miRNAs. Methods: HUVECs were treated by ox-LDL to establish apoptotic model in vitro. miRNA microarrays were employed to detect the expression profile of miRNA in apoptotic HUVECs, and the microarray results were validated by quantitative real-time PCR. The miRNA-targeted genes were predicted by online-available software. The gene ontology (GO) database and KEGG pathway database were used to determine the functions of these target genes. Results: With the use of a microarray, we found that ox-LDL up-regulated four miRNAs (hsa-miR-142-3p, hsa-miR-365, hsa-let-7c, hsa-miR-1207-3p) as well as down-regulated eleven miRNAs (hsa-miR-32, hsa-miR-589, hsa-miR-18b, hsa-miR-429, hsa-miR-155, hsa-miR-590-5p, hsa-miR-1197, hsa-miR-222, hsa-miR-374a, hsa-miR-33a, hsa-miR-375) in HUVECs. The expression levels of hsa-miR-142-3p, hsa-miR-365, hsa-miR-590-5p and hsa-miR-33a were validated in accordance with the results of real-time PCR (qRT-PCR). The bioinformatics analysis indicated that the potential target genes of these aberrantly expressed miRNAs participate in the regulation of cell proliferation, apoptosis, metabolism and oncology gene expression. Conclusion: miRNA expression profile in apoptotic HUVECs induced by ox-LDL has significantly changes, which may contribute to endothelial dysfunction and development of atherosclerosis.