Analysis of cardiovascular disease-related NF-κB-regulated genes and microRNAs in TNFα-treated primary mouse vascular endothelial cells

Abstract

Activated nuclear factor-κB (NF-κB) plays an important role in the development of cardiovascular disease (CVD) through its regulated genes and microRNAs (miRNAs). However, the gene regulation profile remains unclear. In this study, primary mouse vascular endothelial cells (pMVECs) were employed to detect CVD-related NF-κB-regulated genes and miRNAs. Genechip assay identified 77 NF-κB-regulated genes, including 45 upregulated and 32 downregulated genes, in tumor necrosis factor α (TNFα)-treated pMVECs. Ten of these genes were also found to be regulated by NF-κB in TNFα-treated HeLa cells. Quantitative real-time PCR (qRT-PCR) assay confirmed the upregulation of Egr1, Tnf, and Btg2 by NF-κB in the TNFα-treated pMVECs. The functional annotation revealed that many NF-κB-regulated genes identified in pMVECs were clustered into classical NF-κB-involved biological processes. Genechip assay also identified 26 NF-κB-regulated miRNAs, of which 21 were upregulated and 5 downregulated, in the TNFα-treated pMVECs. Further analysis showed that nine of the identified genes are regulated by seven of these miRNAs. Finally, among the identified NF-κB-regulated genes and miRNAs, 5 genes and 12 miRNAs were associated with CVD by miRWalk and genetic association database analysis. Taken together, these findings show an intricate gene regulation network raised by NF-κB in TNFα-treated pMVECs. The network provides new insights for understanding the molecular mechanism underlying the progression of CVD.

概要

目 的

鉴定与心血管疾病相关的核因子 κB(NF-κB)调控的基因和小 RNA (microRNA), 探讨疾病发生发展的机制。

创新点

构建心血管疾病相关的NF-κB 调控网络。

方 法

基于 NF-κB 转录活性差异的小鼠原代血管内皮细胞模型, 采用基因芯片 (Genechip) 检测 NF-κB 调控的基因和 microRNA。 再通过实时荧光定量聚合酶链反应 (qRT-PCR) 和生物信息学方法进行差异基因和 microRNA 的筛选、 验证、 功能注释, 从而发现与心血管疾病相关的 NF-κB 调控的基因和 microRNA。

结 论

在肿瘤坏死因子 α (TNFα) 处理的小鼠原代血管内皮细胞中: NF-κB 调控 77 个基因, 其中 45 个基因上调, 32 个基因下调。 NF-κB 还在 TNFα 处理的 HeLa 细胞中调控其中 10 个基因。 通过 qRT-PCR 验证了 NF-κB 上调 Egr1、 Tnf 和 Btg2 的表达。 基因功能注释表明, 许多 NF-κB 调节的基因聚类到经典的 NF-κB 参与的生物学过程。 在 TNFα 处理的小鼠原代血管内皮细胞中还发现: NF-κB 调控 26 个 microRNA, 其中 21 个上调, 5 个下调。 进一步研究发现, 7 个 NF-κB 调控的 microRNA 还可能调控 9 个 NF-κB 调控的基因。 最后通过检索数据库发现, 5 个 NF-κB 调控的基因和 12 个 NF-κB 调控的 microRNA 与心血管疾病相关。 因此, 本研究提升了对心血管疾病进展分子机制的理解。