miR-106b-5p在调节内皮细胞基因表达谱中的作用

Role of miR-106b-5p in the regulation of gene profiles in endothelial cells

目的:评估miR-106b-5p对内皮细胞基因表达谱的影响。方法:对动脉粥样硬化组(n=9)及对照组(n=9)血浆中miRNAs表达谱进行筛查,采用共表达网络分别对两组样本全体miRNAs的共表达模式进行分析。选取从两组网络中得到的共表达地位差异最为显著的miR-106b-5p进一步研究,通过转染miR-106b-5p mimics上调人脐静脉内皮细胞的miR-106b-5p表达水平,筛查转染后差异基因表达谱,并进一步通过日本京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)信号转导通路数据库对差异基因富集的信号通路进行分析。结果:动脉粥样硬化组患者血浆中miRNAs的共表达模式(140个节点,1 154条连接线)与对照组(140个节点,612条连接线)相比存在明显差异,在过表达miR-106b-5p后人脐静脉内皮细胞有746个基因水平发生了显著变化(组间差异倍数≥1.5,芯片错误发现率<0.01),主要包括磷脂酰肌醇-3激酶(phosphoinositide 3-kinase,PI3K)/蛋白激酶B(protein kinase B,PKB,又称Akt)信号通路、哺乳动物雷帕霉素受体蛋白(mammalian target of rapamycin,m TOR)信号通路、转化生长因子-β(transforming growth factor-β,TGF-β)信号通路、酪氨酸激酶-信号转导及转录激活因子信号通路(janus kinase/signal transducer and activator of transcription,Jak-STAT)信号通路、肿瘤坏死因子(tumor necrosis factor,TNF)信号通路、toll样受体(toll-like receptor,TLR)信号通路、血管内皮生长因子(vascular endothelial growth factor,VEGF)信号通路等20个信号通路。结论:动脉粥样硬化患者血浆中miRNAs共表达模式发生了显著变化,其中共表达地位差异最为显著的miR-106b-5p可靶向调节血管内皮细胞多个信号通路。

Objective: To evaluate the role of miR-106b-5p in the regulation of gene expression in endothelial cells. Methods: The Taqman low-density microRNAs (miRNAs) array (TLDA) was used to identify miRNA expression profiles in the plasma of patients with atherosclerotic coronary artery disease (CAD)(atherosclerosis group, n =9) and individuals without atherosclerotic CAD disease (control group, n =9). A weighed and undirected miRNA coexpression network analysis was performed to investigate the interactions among miRNAs in the two groups. MiR-106b-5p, whose coexpression pattern in atherosclerosis group was most different from that of control group, was further studied. Human umbilical vein endothelial cells (HUVEC) were transfected with miR-106b-5p mimic or negative control mimic, and Affymetrix GeneChip Human Transcriptome Array 2.0 was used to screen the differential gene expression profiles after transfection. And the signal transduction pathway of differential gene profiles was further analyzed in Kyoto Encyclopedia of Genes and Genomes (KEGG) signal pathway database. After parsing the whole KEGG database, all differentially expressed genes involved pathways were extracted, and the hypergeometric distribution was used to calculate the pathway enrichment. Results: The coexpression pattern of the patients with atherosclerosis (140 nodes, 1 154 edges) differed from that of the non-atherosclerosis control group (140 nodes, 612 edges). The analysis of array data with significant analysis of microarray (SAM) identified 746 significantly deregulated genes (fold change ≥ 1.5 and false discovery rate < 0.01) altered by overexpression of miR-106b-5p with miR-106b-5p mimic in HUVEC. By calculating the pathway enrichment, we found that multiple signaling pathways enriched in differential gene profiles were closely related to the process of formation and rupture of atherosclerotic plaque, including phosphatidylinositol-3 kinase ( PI3K )/ protein kinase B ( PKB , also called Akt), mammalian target of rapamycin ( mTOR ), transforming growth factor-β( TGF-β), janus kinase / signal transducer and activator of transcription ( Jak-STAT ), tumor necrosis factor ( TNF ), toll like receptor ( TLR ) and hypoxia-inducible factor 1α( HIF-1α) and other signal pathways. Conclusion: The coexpression pattern of miRNAs in plasma of patients with atherosclerosis is more significantly changed than that of individuals without atherosclerotic disease. MiR-106b-5p, which shows the most significant dif- ference between groups, targets multiple signal pathways in vascular endothelial cells, and might play an important role in the regulatory network of atherosclerotic gene expression.